Transfer of rol genes and Evaluation of
Artemisinin Synthesis in Transgenic Artemisia
annua L. and Artemisia dubia Wall.
By
BUSHRA HAFEEZ KIANI
Department of Biochemistry
Quaid-i-Azam University
Islamabad, Pakistan
2012
Transfer of rol genes and Evaluation of
Artemisinin Synthesis in Transgenic Artemisia
annua L. and Artemisia dubia Wall.
Submitted by
BUSHRA HAFEEZ KIANI
Thesis Submitted to
Department of Biochemistry
Quaid-i-Azam University, Islamabad
In the partial fulfillment of the requirements for the degree of
Doctor of Philosophy
in
Biochemistry
Department of Biochemistry
Quaid-i-Azam University
Islamabad, Pakistan
2012
In the name of ALLAH who is the Beneficent and The
most Merciful, Guardian of faith, The Majestic,
The Bestower and The Forgiver,
Whose help and guidance
I always importune at every step.
CERTIFICATE
The department of Biochemistry, Quaid-i-Azam University, Islamabad accepts this
thesis submitted by Bushra Hafeez Kiani in its present form, as satisfying the thesis
requirement for the Degree of Doctor of Philosophy (Ph.D.) in Biochemistry and
Molecular Biology.
Supervisor: _________________
Prof. Dr. Bushra Mirza
External Examiner: _________________
External Examiner: _________________
Chairman: _________________
Prof. Dr. Bushra Mirza
Dated: __________________
Declaration
I hereby declare that the work presented in this thesis is my own effort except where
others acknowledged and that the thesis is my own composition. No part of this thesis
has previously been presented for any other degree.
Bushra Hafeez Kiani
DEDICATION
This humble effort is sincerely dedicated to
My Abbu Ji whose wishes and devotions
Made me capable of achieving such a Success
and to my sweet, ever-loving Ammi Ji
whose hands always rose for my Success,
And
My Family
(Words are futile to that
They have done for me)
Contents
Acknowledgements
i
List of Tables
iii
List of Figures
iv
List of Abbreviations
x
Abstract
xiii
Chapter 1
Introduction and review of Literature
1
1.1
Genus Artemisia, a natural source of artemisinin
2
1.1.1
Evidence of presence of artemisinin in some Artemisia
species other than A. annua
2
1.1.2
Evidence of antimalarial activity of some Artemisia species
other than A. annua
3
1.1.3
Taxonomic classification of genus Artemisia
3
1.1.4
History of Artemisia
4
1.1.5
Morphology
4
1.1.6
Distribution
6
1.2
Morphology of Artemisia annua
6
1.3
Morphology of Artemisia dubia
8
1.3.1
Selection of Artemisia dubia for artemisinin enhancement
9
1.4
Secondary metabolites and Medicinal Constituents of
Artemisia
10
1.5
Medicinal Importance of genus Artemisia
11
1.5.1
Non medicinal importance of the genus Artemisia
11
1.5.2
Toxic effects of Artemisia
12
1.6
Artemisinin
12
1.6.1
Artemisinin Synthesis and Storage
13
1.6.2
Derivatives of artemisinin
14
1.6.3
Mode of action of artemisinin
15
1.6.4
Biological activities of artemisinin
16
1.6.4.1
Malaria
17
1.6.4.2
Schistosomiasis
18
1.6.4.3
Hepatitis B
18
1.6.4.4
Cancer
19
1.6.4.4.1
Effect of artemisinin and its derivatives on different types
of cancer
19
1.6.4.5
Herbicides
20
1.7
Improvement of Artemisinin Content
20
1.7.1
Selection of best cultivar
21
1.7.2
Selection of different stages
21
1.7.3
Tissue Culture of Artemisia
22
1.7.4
Transformation
24
1.7.4.1
Agrobacterium-mediated Transformation
24
1.7.4.1.1
Classification of Agrobacterium
24
1.7.4.1.2
Agrobacterium tumefaciens
24
1.7.4.1.3
Agrobacterium rhizogenes
25
1.7.4.2
Molecular mechanism of Agrobacterium tumefacienes
mediated transformation
26
1.7.4.2.1
The binary vector strategy
28
1.7.4.2.2
The cointegration strategy
28
1.7.4.3
Production of transformed plants with Ti plasmid
29
1.7.4.4
Mechanism of A. rhizogenes infection
30
1.8
Agrobacterium tumefacienes mediated transformation in
Artemisia species
31
1.9
Agrobacterium rhizogenes mediated transformation in
Artemisia species
32
1.10
Role of rol genes in Plants
33
1.10.1
Synergistic effects of rol genes
37
1.11
Extraction of artemisinin
38
1.12
Analysis of artemisinin
39
1.13
Biosynthetic pathway of artemisinin
40
1.13.1
Post-IPP terpene biosynthesis
41
1.13.2
Committed steps in artemisinin biosynthesis
42
1.13.3
Regulation of the artemisinin biosynthetic pathway
44
1.14
Aims and objective
46
Chapter 2
Genetic Transformation of Artemisia annua and
47
Artemisia
with
dubia
Agrobacterium
rol
tumefacienes
ABC
and
genes
through
Agrobacterium
rhizogenes
2.1
Introduction
47
2.2
Materials and methods
48
2.2.1
Glassware and chemicals
48
2.2.1.2
Medium
48
2.2.3
Inoculation area and manipulation tools
48
2.2.4
Culturing of tissues
49
2.2.5
Collection of Plant Material
49
2.2.6
Seed Germination
49
2.2.7
Culture environment
49
2.2.8
Transformation of Artemisia species with rol ABC genes
50
2.2.8.1
Agrobacterium tumefaciens mediated transformation of
Artemisia dubia and Artemisia annua with rol ABC Genes
50
2.2.8.2
Plant material
50
2.2.8.3
Bacterial strain and plasmid construction
50
2.2.9
Transformation procedure
51
2.2.9.1
Preparation of Explants
51
2.2.10
Co cultivation
51
2.2.10.1
Co cultivation medium
51
2.2.10.2
Co cultivation
51
2.2.11
Selection and regeneration
52
2.2.12
Rooting
52
2.2.13
Transplantations to pots and acclimatization
53
2.2.14
53
2.2.15
Agrobacterium rhizogenes mediated transformation of
Artemisia dubia and Artemisia annua with rol ABC Genes
In-vitro plant production and sterilization
2.2.16
2.2.16.1
Media used for establishment of hairy root cultures
Preparation of MS shooting medium
54
54
2.2.16.2
Preparation of ½MS rooting medium
54
2.2.16.3
Preparation of nutrient agar medium
54
2.2.16.4
Preparation of B5 solid medium
54
2.2.16.5
Preparation of B5 selection medium
54
2.2.16.6
Preparation of B5 liquid medium
54
2.2.17
Induction of Hairy Roots
55
2.2.17.1
Preparation of A. rhizogenes strain
55
2.2.17.2
55
2.2.17.3
A. rhizogenes infection on Artemisia annua and Artemisia
dubia
Transfer of hairy root cultures on B5 solid medium
2.2.17.4
Transfer of hairy roots in to B5 liquid medium
56
2.2.18
Molecular analysis
56
2.2.18.1
Isolation of genomic DNA from plant leaves
56
2.2.18.1.1
Composition of CTAB buffer
57
2.2.18.2
Extraction and purification of plasmid DNA
57
53
55
62.2.18.2.1
Solutions
58
2.2.19
Polymerase chain reaction
58
2.2.19.1
Primers used during PCR
59
2.2.19.2
Agarose gel electrophoresis
59
2.2.20
Southern blot analysis
59
2.2.20.1
DNA restriction
60
2.2.20.2
Agarose gel electrophoresis
60
2.2.20.3
Transfer of restriction fragments to membrane
60
2.2.20.4
Labeling of DNA
60
2.2.20.5
Hybridization process
61
2.3
Results
61
2.3.2
Seed Surface Sterilization
61
2.3.3
Medium for Seed Germination
62
2.3.4
Types of explants
65
2.3.5
Transformation
66
2.3.5.1
Effect of Co-cultivation Period on Transformation
66
2.3.5.2
Regeneration of Transgenic Plants
66
2.3.5.2.1
Effect of Antibiotics on Agrobacterium in Regeneration
Medium
66
2.3.5.2.2
Regeneration of Transgenic Plants
66
2.3.5.3
Rooting of Transformed Plants
71
2.3.6
Morphological Analysis of Transformed Plants
72
2.3.7
In-vitro plant production and sterilization
74
2.3.8
Production of Hairy Roots
75
2.3.9
Molecular Analysis of Transformed Plants
75
2.3.9.1
Genomic DNA Extraction
75
2.3.9.2
Polymerase Chain Reaction (PCR)
77
2.3.10
Southern Blotting
79
2.3.11
Acclimatization
81
2.4
Conclusion
83
Chapter 3
Analysis of Artemisinin and its Derivatives and their
comparison in transformed and un-transformed plants
of Artemisia annua and Artemisia dubia
3.1
Introduction
84
3.2
Materials and methods
85
3.2.1
Extraction of artemisinin
85
3.2.2
Reagents used
85
3.2.2.1
Solvents used
85
3.2.2.1.1
Solvent No.1: Mixture of Ethyl acetate and Hexane in the
ratio of 5:95
85
86
3.2.2.1.3
Solvent No.2: Mixture of Acetonitrile and Water in the ratio
of 50:50
Biomass and Solvent Ratio
3.2.3
Analysis of artemisinin content
86
3.2.3.1
High performance liquid chromatography
86
3.2.3.1.1
Preparation of mobile phase
86
3.2.3.1.2
Preparation of dilutions of standard
87
3.2.3.1.3
Column used for HPLC
87
3.2.3.1.4
Flow of mobile phase through HPLC system
87
3.2.3.1.5
Detector
87
3.2.3.1.6
Retention time of peak
87
3.2.3.1.7
Injection volume
87
3.2.3.1.8
Calibration curve
88
3.2.3.1.9
Quantification of artemisinin and its derivatives in sample
88
3.2.3.1.10
Verification of artemisinin and derivatives of artemisinin
88
3.2.2.1.2
86
concentrations
3.2.4
Statistical analysis
89
3.3
Results
89
3.3.1
Analysis of artemisinin content in transformed and
untransformed Artemisia annua and Artemisia dubia
89
3.3.2
Analysis of derivatives of artemisinin in transformed and
untransformed Artemisia annua and Artemisia dubia plants
93
concentrations
3.3.3
Statistical Analysis
108
3.3.3.1
Comparative analysis of artemisinin and its derivatives in
different tissues of Artemisia annua and Artemisia dubia
108
3.4
Conclusion
109
Chapter 4
Analysis
of
Metabolic
pathway
and
Trichome
development and their comparison in transformed and
un-transformed plants of Artemisia annua and Artemisia
dubia
4.1
Introduction
4.2
Materials and methods
111
112
112
4.2.1
Analysis of Metabolic Pathway
4.2.1.1
RNA Extraction
112
4.2.1.2
cDNA synthesis
113
4.2.2
Quantitative Real Time Polymerase chain reaction (qRT-
115
PCR)
4.2.3
Analysis of Trichome density
116
4.2.3.1
Environmental Scanning Electron Microscope (ESEM)
117
setup
4.3
Results
117
4.3.1
Analysis of Metabolic pathway and Trichome development
117
4.3.2
Relative expression of genes involved in metabolic pathway
117
of artemisinin biosynthesis
4.3.3
Trichome Development
119
4.3.4
Statistical Analysis
122
4.3.4.1
Analysis of different genes involved in artemisinin
122
production pathway in different tissues of Artemisia annua
and Artemisia dubia
4.3.4
Analysis of Trichome density
125
4.4
Conclusion
131
Chapter 5
Analysis of anticancer activity on Breast Cancer Cell
lines
and
comparison
in
transformed
and
untransformed Artemisia annua and Artemisia dubia
5.1
Introduction
133
5.2
Materials and methods
134
5.2.1
Analysis of transformed and untransformed A.annua and
A.dubia on Breast Cancer Cell lines
134
5.2.2
Sample preparation
134
5.2.2.1
Hexane extraction
134
5.2.2.2
Aqueous extraction
134
5.2.3
Cell Preparation
135
5.2.3.1
Cells
135
5.2.3.2
Cell inoculation
135
5.2.3.3
Fixation protocol
135
5.2.4
Sulforhodamine B (SRB) Antiproliferative Assay
136
5.3
Results
136
5.3.1
Analysis of transformed and untransformed Artemisia
136
annua and Artemisia dubia on Breast Cancer Cell lines.
5.4
Conclusion
143
Chapter 6
Sequence analysis of Artemisia annua and Artemisia
dubia
and
comparison
in
transformed
and
untransformed plants
6.1
Introduction
144
6.2
Materials and methods
145
6.2.1
Sequence analysis of transformed and untransformed
A.annua and A.dubia
145
6.2.2
Purification and Fragmentation of mRNA
145
6.2.2.1
Formation and purification of RBP
145
6.2.2.2
Formation and purification of RFP
146
6.2.3
Synthesis of First Strand cDNA
148
6.2.3.1
Formation of CDP
148
4.2.4
Synthesis of Second Strand cDNA
149
4.2.4.1
Addition of SSM
149
6.2.5
End Repairing
150
6.2.5.1
Formation of IMP
150
6.2.6
3' Ends Adenylation
151
6.2.6.1
Addition of ATL
152
6.2.7
Ligate Adapters
152
6.2.7.1
Addition of LIG
152
6.2.8
DNA Fragments Enrichment
155
6.2.8.1
PCR reaction
155
6.2.8.2
Amplification of PCR plates
155
6.2.8.3
Cleaning of PCR plates
156
6.2.9
Validation of Libraries
157
6.2.9.1
Quantification of Libraries
157
6.2.9.2
Quality Control
157
6.2.9.2
Evaluation of sequence homology
157
6.3
Results
157
6.4.1
Comparison of various Transgenic Lines with respect to
158
specific Contigs
6.4.2
Contigs of A.annua and A.dubia producing more than 90%
168
homology
6.4.4
Contigs of A.annua and A.dubia producing 80-85%
202
homology
Contigs of A.annua and A.dubia do not produce homology
202
6.5
Conclusion
203
Chapter 7
Discussion
7.1
Agrobacterium tumefaciens mediated transformation of
6.4.5
205
Artemisia dubia and Artemisia annua with rol ABC Genes
7.2
Agrobacterium rhizogenes mediated transformation of
210
Artemisia dubia and Artemisia annua with rol ABC Genes
7.3
Analysis of Artemisinin and its Derivatives
212
7.4
Analysis of Metabolic pathway and Trichome development
218
7.5
Analysis of transformed and untransformed Artemisia
220
annua and Artemisia dubia on Breast Cancer Cell lines
7.6
Sequence analysis of Artemisia annua and Artemisia dubia
223
and comparison in transformed and untransformed plants
Conclusion
227
Future strategies
228
References
229
Publications
272
Appendices
273
ACKNOWLEDGEMENT
I offer my humblest thanks to ALMIGHTY ALLAH the Lord of the
creation, the Merciful and the source of all knowledge and wisdom and to His
Prophet Hazrat Muhammad (S.A.W) who guided mankind to get out of
darkness of illiteracy and ignorance.
I am lacking with the words, to express my extreme profound
appreciation and sincere thanks to my Supervisor and Chairperson,
Department of Biochemistry, Quaid-i-Azam University Islamabad Professor
Dr. Bushra Mirza, for her guidance, precious advices, genuine interest,
constructive criticism, thoughtful suggestions and generous supervision
throughout my research work. This tedious task would not have been possible
without her co-operation, kind interest and moral encouragement.
I am extremely grateful to Dr. Guy C Barker, Professor and Director of
Genomics, University of Warwick, UK for providing research facilities to
carry out my research project in his laboratory and also for his cooperation,
useful suggestions, inspiration, reassurance, guidance and counseling from
time to time to complete this research project.
It is my pleasure to mention John S, Irnia N, Linda B, Dr, Raja AA, Dr.
Vickey, Dr. Babar M, Dr. Kapila P, Dr Sajjad A, Alison J, Sara T, Anna M,
Beyony K, Tina M, Rachel M, for their great cooperation and support during
my stay at Wellesbourne, UK. I believe that without their help my Ph.D
project may not have been furnished so quickly and nicely. I am also
extremely grateful to Isolda R, Ph.D student in Chemistry Department,
University of Warwick UK for her kind cooperation during analysis of my
samples on Cancer Cell Lines. I am also thankful to Mr. Martin Davis,
Research Assitant, Department of Engneering, University of Warwick UK,
for his kind cooperation to provide the facility for ESEM.
I would like to extend my deepest appreciation to those people, who
helped me in one way or another during my stay at Department of
Biochemistry, QAU Islamabad, Pakistan. During my PhD research, I worked
with a great number of people; I wish to convey my gratitude to all of them
specially, Dr. Abdul Mannan, Dr. Ihsan.UL.Haq, Dr. Sheeraz Ahmad, Dr.
Waseem Ahmad, Dr. Waheed Ahmad, Dr. Nazif Ullah, Aman Ullah, Waqas
Kiani, Tanveer Ahmad, Dr. Naila, Dr. Maryam, Samreen, Laila, Rehana,
Nosheen, Samiya, Saira, Maryam and Tooba.
I would like to pay cordial thanks to Dr. Naila Safdar for her
guidance, Cooperation and care which helped me put up with all the
frustrations encountered during my research work. I will always remain
thankful to my dearest friends Amama, Fari, Rehana, Rizwana, Humaira,
Sheerin, Jasia, Nosheen, Irnia for their moral support and help. I will always
remember the cooperation and help of Irshad N, Amir K, Tariq A, and Murad.
I am thankful to Higher Education Commission (HEC) Pakistan for
providing me indigenous fellowship and IRSIP scholarship. I believe that HEC
funding added to my strength a lot.
I am also grateful to my Mamu Dr.Amjad Hussain Tariq Kiani and My
brother in-law Khaleeq Kiani and Imran Kiani for their encouragement and
inspiring guidance in successful completion of this work.
I am very thankful to my nephews Nehal, Minaam, Izaan and my nieces
Maham and Zoona for their sweet and tension releasing attitude. I will never
forget their innocent love for me.
My special thanks are reserve for my family for making me what I am
today, how can I forget my loving brother, Dr.Qamar Kiani, Sweet Shumaila
bhabi, my sweet sisters Noshi baji, Fozi baji and Sobi baji whose prayers are
a source of unmatching support for me.
Last but not the least, I owe my deepest gratitude to my loving
parents whose invaluable prayers and embolding attitude kept my spirit high
to strive for the knowledge and integrity which enabled me to reach the
milestone (Allah Bless them always, Aameen).
I would like to express my apology to those who ever had a soft corner
for me but I missed to mention them personally.
Finally, all the errors that remains, are mine, alone.
Bushra Hafeez Kiani
List of Tables
No.
Title
Page no.
Table 2.1
Effect of medium on germination of seeds
62
Table 2.2
Morphological differences observed among Transgenic and
73
Control Plants
Table 3.1
Analysis of Variance Table for factors affecting Production
108
of Artemisinin and its Derivatives in different tissues of
A.annua and A.dubia
Table 4.1
Different components used for cDNA Synthesis
114
Table 4.2
Different components used for Master Mix for cDNA
114
Synthesis
Table 4.3
Primers used for qRT-PCR
116
Table 4.4
Analysis of Variance Table for factors affecting different
123
genes involved in artemisinin production pathway in
different tissues of A.annua and A.dubia
Table 4.5
Analysis of Variance for significant difference between
126
different species of Artemisia plant
Table 6.1
Comparison of various Transgenic Lines with respect to
159
specific Contigs
Table 6.2
Sequences producing Significant Homology
169
List of Figures
No
Title
Page No
Fig 1.1
Malaria Distribution and Statistics
1
Fig.1.2
Floral morphology of Artemisia (A) A daisy flower
5
(Asteraceae) – a composite head of many small flowers (B)
Detailed description of floral parts.
Fig 1.3
Artemisia annua
7
Fig 1.4
Artemisia dubia
8
Fig 1.5
Structure of Artemisinin
12
Fig 1.6
Chemical structure of artemisinin and its derivatives
15
Fig 1.7
Mechanism of the reductive activation of artemisinin by iron
16
(II)-heme, leading to covalent heme-artemisinin adducts.
Fig 1.8
Diagramic
representation
of
biological
activities
of
17
artemisinin
Fig 1.9
Structure of the Ti-plasmid of A.tumefacienes
25
Fig 1.10
Structure of the Ri-plasmid of A.rhizogenes
26
Fig 1.11
Transformation of plant cell by Agrobacterium tumefacienes
27
harbouring a wild-type Ti plasmid
Fig 1.12
The binary vector strategy. Plasmids A and B complement
28
each other when present together in the same A. tumefaciens
cell. The T-DNA carried by plasmid B is transferred to the
plant chromosomal DNA by proteins coded by genes carried
by plasmid A.
Fig 1.13
The cointegration strategy
29
Fig 1.14
Process of hairy roots production through Agrobacterium
31
rhizogenes infection
Fig 1.15
Early biosynthetic pathway for artemisinin: steps in the
41
MVA and MEP pathways and post-IPP steps
Fig 1.16
Biosynthetic pathway of artemisinin
44
Fig 2.1
Schematic diagram of the T-DNA region of the plasmid
50
No
Title
Page No
pRT99
Fig 2.2
Percentage seed germination with different duration of
63
exposure to 0.1% (w/v) Mercuric Chloride
Fig 2.3
In vitro grown seedlings of Artemisia dubia (a, b)
64
and Artemisia annua (c, d) on MS.
Fig 2.4
Leaf and Stem Explants used for transformation Artemisia
65
dubia (a, b) and Artemisia annua (c, d)
Fig2.5(a)
Regeneration response of Artemisia dubia explants on
67
selection media after incubation in Agrobacterium solution
for various durations
Fig2.5(b)
Regeneration response of Artemisia annua explants on
selection
68
media after incubation in Agrobacterium
solution for various durations
Fig 2.6
Effect of incubation period in Agrobacterium solution
69
on regeneration efficiency
Fig 2.7
Shooting of transformants from different explants
70
Fig 2.8
Rooting response of transformed plants
72
Artemisia dubia (a, b) and Artemisia annua (c, d)
Fig 2.9
Comparison of Transformed and Non-transformed plants
74
Fig 2.10
In-vitro grown plantlet of (a) Artemisia dubia (b) Artemisia
75
annua
Fig 2.11
Induction of hairy roots from stems infected with
76
Agrobacterium rhizogenes
Fig2.12(a) Amplified product of transgenic Artemisia dubia
78
Fig2.12(b) Amplified product of transgenic Artemisia annua
79
Fig2.13
80
Southern blot analysis (a) Artemisia dubia (b) Artemisia
annua
Fig 2.14
Transformed plant in green house
82
Fig 3.1
HPLC Chromatograms of (a) Standard Artemisinin (b)
90
Artemisia annua Sample (A2) (c) Spike Sample (A.annua
No
Title
Page No
sample + standard Artemisinin)
Fig 3.2
HPLC Chromatograms of (a) Standard Artemisinin (b)
91
Artemisia dubia Sample (D2) (c) Spike Sample (A.dubia
sample + standard Artemisinin)
Fig 3.3
Comparative analysis of Artemisinin content in shoots, roots
92
and hairy roots of transformed and un-transformed plants of
Artemisia dubia and Artemisia annua. The average of three
plants is shown together with error bars showing SE.
A2+Artemisinin and D2+ Artemisinin are the spike samples
used for verification of artemisinin concentration.
Fig 3.4
HPLC Chromatograms of (a) Standard Artemether (b)
95
Artemisia annua Sample (A2) (c) Spike Sample (A.annua
sample + standard Artemether)
Fig 3.5
HPLC Chromatograms of (a) Standard Artemether (b)
96
Artemisia dubia Sample (D1) (c) Spike Sample (A.dubia
sample + standard Artemether)
Fig 3.6
HPLC Chromatograms of (a) Standard Arteether (b)
97
Artemisia annua Sample (A2) (c) Spike Sample (A.annua
sample + standard Arteether)
Fig 3.7
HPLC Chromatograms of (a) Standard Arteether (b)
98
Artemisia dubia Sample (D1) (c) Spike Sample (A.dubia
sample + standard Arteether)
Fig 3.8
HPLC Chromatograms of (a) Standard Dihydroartemisinin
99
(b) Artemisia annua Sample (A2) (c) Spike Sample (A.annua
sample + standard Dihydroartemisinin)
Fig 3.9
HPLC Chromatograms of (a) Standard Dihydroartemisinin
100
(b) Artemisia dubia Sample (D1) (c) Spike Sample (A.dubia
sample + standard Dihydroartemisinin)
Fig 3.10
HPLC Chromatograms of (a) Standard Artesunate (b)
Artemisia annua Sample (A2) (c) Spike Sample (A.annua
sample + standard Artesunate)
101
No
Fig 3.11
Title
Page No
HPLC Chromatograms of (a) Standard Artesunate (b)
102
Artemisia dubia Sample (D1) (c) Spike Sample (A.dubia
sample + standard Artesunate)
Fig 3.12
Artemether content in shoots roots and hairy roots of
103
A.annua.A1, A2 and A3 represents leaf samples from three
different transgenic lines, AR1, AR2 and AR3 represents
roots of transgenic lines A1, A2 and A3 respectively, AH1
and AH2 represents two transgenic lines of hairy roots, AC
and ARC represent control shoots and roots. A2+Artemether
and D2+ Artemether are the spike samples used for
verification of artemether concentration.
Fig 3.13
Arteether content in shoots roots and hairy roots of A.annua.
104
A1, A2 and A3 represents leaf samples from three different
transgenic lines, AR1, AR2 and AR3 represents roots of
transgenic lines A1, A2 and A3 respectively, AH1 and AH2
represents two transgenic lines of hairy roots, AC and ARC
represent control shoots and roots. A2+Arteether and D2+
Arteether are the spike samples used for verification of
arteether concentration.
Fig 3.14
Dihydro-artemisinin content in shoots roots and hairy roots
105
of A.annua.A1, A2 and A3 represents leaf samples from
three different transgenic lines, AR1, AR2 and AR3
represents roots of transgenic lines A1, A2 and A3
respectively, AH1 and AH2 represents two transgenic lines
of hairy roots, AC and ARC represent control shoots and
roots. A2+Dihydroartemisinin and D2+ Dihydroartemisinin
are
the
spike
samples
used
for
verification
of
dihydroartemisinin concentration.
Fig 3.15
Artesunate content in shoots roots and hairy roots of
A.annua.A1, A2 and A3 represents leaf samples from three
106
No
Title
Page No
different transgenic lines, AR1, AR2 and AR3 represents
roots of transgenic lines A1, A2 and A3 respectively, AH1
and AH2 represents two transgenic lines of hairy roots, AC
and ARC represent control shoots and roots. A2+Arteether
and D2+ Arteether are the spike samples used for verification
of arteether concentration.
Fig 3.16
Comparative and Statistical analysis of Average Artemisinin,
107
Artemether, Arteether, Dihydro-artemisinin and Artesunate
content in shoots, roots and hairy roots of transformed and
un-transformed plants of A.dubia and A.annua. Each value is
the mean of three replicates. Any two means having a
common alphabet are not significantly different at p = 0.05
using LSD. Vertical bar represents the standard error of the 3
means.
Fig 4.1(A) Expression of genes in leaves of A.annua.
119
Fig 4.1(B) Expression of genes in hairy roots, roots of transformed
120
shoots of A.annua.
Fig 4.1(C) Expression of genes in leaves of A.dubia.
121
Fig 4.1(D) Expression of genes in hairy roots, roots of transformed
122
shoots of A.dubia.
Fig 4.2
Statistical and comparative analysis of production of
125
artemisinin and its derivatives in different tissues of
Artemisia annua and Artemisia dubia. Each value is the
mean of three replicates. Any two means having a common
alphabet are not significantly different at p = 0.05 using LSD.
Vertical bar represents the standard error of the 3 means
Fig 4.3(A) Comparative analysis of trichome density in shoots roots and
127
hairy roots of A.annua and A.dubia
Fig 4.3(B) Comparison of trichome density in different tissues of
transformed and untransformed plants of A.annua and
A.dubia
130
No
Title
Page No
Fig 5.1(A) Analysis of percentage survival in hexane fractions of
139
transformed and untransformed plants, roots and hairy roots
of A.annua and A.dubia at 50 µg concentration. The IC50
values are shown as values above the bars. *Artemisinin (IC
50
= 0.21 µM) was used as positive control
Fig 5.1(B) Analysis of percentage survival in aqueous fractions of
140
transformed and untransformed plants, roots and hairy roots
of A.annua and A.dubia at 50 µg concentration. The IC50
values are shown as values above the bars. *Artemisinin (IC
50
= 0.21 µM) was used as positive control
Fig 5.1(C) Comparative analysis of average percentage inhibition in
hexane
and
aqueous
fractions
of
transformed
141
and
untransformed plants, roots and hairy roots of A.annua and
A.dubia at 50 µg concentration. The IC50 values are shown as
values above the bars. *Artemisinin (IC
50
= 0.21 µM) was
used as positive control
Fig 5.2
Comparative
correlation
analysis
between
artemisinin
content and anticancer activity in hexane and aqueous
fractions of transformed and untransformed plants, roots and
hairy roots of A.annua and A.dubia. The R2 values are shown
as values above the bars. Those samples that had R2 value
0.6-0.9 were significantly correlated. Data was analyzed for
significant correlation by using Graph Pad Prism Method.
142
List of Abbreviations
ACT
Artemisinin-based Combination Therapy
ADS
Amorpha-4,11-Diene Synthase
AIDS
Acquired Immune Deficiency Syndrome
ANOVA
Analysis of Variance
APX
Ascorbate Peroxidase
AsA
L-ascorbic Acid
ATCC
American Type Culture Collection
A.
Artemisia
A. annua
Artemisia annua
A. radiobacter
Agrobacterium radiobacter
A. rhizogenes
Agrobacterium rhizogenes
A. rubi
Agrobacterium rubi
A. tumefaciens
Agrobacterium tumefaciens
A. vitis
Agrobacterium vitis
Bp
Base Pair
cDNA
Complementary Deoxyribonucleic Acid
CE-UV
Capillary Electrophoresis Coupled to UV Detector
Cs
Beta-Caryophyllene Synthase
CYP
Cytochrome P450 CYP71AV1
CYP71AV1
Cytochrome P450 CYP71AV1
DAB
3, 3‟-Diaminobenzidine-HCl
DAD
Diode Array Detector
DEPC
Diethylpyrocarbonate
DHAA
Dihydroartemisinic Acid
DMAPP
Dimethylallyl Diphophate
DMSO
Dimethyl Sulfoxide
DNA
Deoxyribonucleic Acid
DNase
Deoxyribonuclease
DXP
1-Deoxy-D-xylulose-5-Phosphate
DXR
1-Deoxy-D-xylulose-5-Phosphate Reductoisomerase.
DXS
1-Deoxy-D-xylulose-5-Phosphate Synthase
DW
Dry Weight
ELISA
Enzyme-Linked Immunosorbent assay
Eps
Epi-Cedrol Synthase
FDP
Farnesyl Diphosphate
FPS
FDP Synthase
Fs
(E)-Beta-Farnesene Synthase
FW
Fresh Weight
GC
Gas Chromatography
G-C
Guanine-Cytosine
GPX
Glutathione Peroxidase
GSH
Glutathione
HMGR
Hydroxymethylglutaryl-CoA Reductase
HMG-CoA
Hydroxymethylglutaryl-CoA
HPLC
High Performance Liquid Chromatography
IPP
Isopentenyl Diphosphate
L
Liter
L-ascorbate
L-Ascorbic Acid
LSD
Least Significant Difference
mAU
Milli Absorbance Units
MEP
Non-Mevalonate Pathway
mL
Milli Liter
mM
Milli Molar
mRNA
Messenger RNA
MS
Mass Spectrometry
MS medium
Murashige and Skoog Medium
MVA
Mevalonic Acid Pathway
NADPH
Nicotinamide Adenosine Dinucleotide Phosphate plus Hydrogen
NADP
Nicotinamide Adenosine Dinucleotide Phosphate
NAD
Nicotinamide Adenine Dinucleotide
NADH
Nicotinamide Adenine Dinucleotide plus Hydrogen
Ng
Nanogram
Nm
Nanometer
PCR
Polymerase Chain Reaction
RIA
Radioimmunoassay
Ri plasmids
Root Inducing Plasmids
RNA
Ribonucleic Acid
ROS
Reactive Oxygen Species
rRNA
Ribosomal RNA
RT-PCR
Reverse Transcriptase PCR
SOD
Superoxide Dismutase
SQS
Squalene Synthase
SQC
Sesquiterpene Cyclase
T-DNA
Transfer DNA
TLC
Thin Layer Chromatography
WHO
World Health Organization
UV
Ultraviolet
μg/mL
Microgram Per Milliliter
½ MS medium
Half Murashige and Skoog medium
μM
Micro Molar
V
Volume
18S
18S Ribosomal Small Subunit
ΔΔCT
Delta-Delta Cycle Threshold
H2O2
Hydrogen Peroxide
2,4-D
2,4-Dichlorophenoxyacetic Acid
%
Percent
ºC
Degree Centigrade
RBP
RNA Bead Plate
SSM
Second Strand Master
ATL
A-Tailing Mix
ALP
Adaptor Ligation Plate
RFP
RNA Fragmentation Plate
CDP
cDNA Plate
ERP
End Repair Mix
IMP
Insert Modification Plate
LIG
Ligation Mix
CAP
Clean-up ALP Plate
Abstract
Abstract
Artemisinin is an important secondary metabolite of Artemisia annua and
Artemisia dubia. It is a major constituent of Artemisia species. Chemically it is an
endoperoxide sesquiterpene lactone. It is a potent antimalarial drug that has also been
proven very effective in treatment of cancer. The rol genes have been known to
enhance production of secondary metabolites in plants, possibly through stimulation
of defense pathway. This study examines the effect of transformation of A.annua and
A.dubia with the rol genes through Agrobacterium tumefaciens and Agrobacterium
rhizogenes. The artemisinin content, trichome density and expression of key genes in
the biosynthetic pathway of artemisinin were measured. Anticancerous activity of
extracts of transformed and untransformed A.annua and A.dubia was also observed
against MCF-7 breast cancer cell lines. Transcriptomic study of transformed and
control A.annua and A. dubia was carried out as well.
A number of factors like type of explants, effect of sterilization and cocultivation period have been reported to affect the efficiency of A.tumefacienes
mediated transformation. LBA4404 strain of A.tumefacienes containing pRT99
plasmid harboring rol ABC genes were used for the A.tumefacienes mediated
transformation. Mercuric chloride 0.1% for 2 minutes showed the best results for seed
surface sterilization giving 95% germination. Leaf and stem were found best explants
for transformation. Explants were infected with bacterial culture for 5 minutes and
cultured on co-cultivation medium (MS medium with 200 µM acetosyringone) for 48
hours. Explants when cultured on selection medium (MS medium containing 0.1mg
/L BAP supplemented with 50mg/ml kanamycin), resulted in the maximum number of
transformants. Regeneration of transgenic shoots was obtained from both stem and
leaf explants on regeneration medium
(MS medium containing 0.1mg /L BAP
supplemented with 20mg/ml kanamycin and 500mg/l cefotaxime). Eighty percent of
the transgenic A.dubia shoots showed rooting response on half MS medium with
0.025mg/L NAA, while transgenic shoots of A.annua produced roots on half MS
medium with 0.1mg/L NAA. Control and transgenic plants were transferred to small
pots and acclimatized. Morphological differences like increased size and broad leaves
were observed. Confirmation of transformation was made through PCR for rol A, B
and C genes. Southern blot analysis was performed to check the copy number of
inserted genes.
A.rhizogenes strain LBA9402 and LBA 8196 carrying rol genes were used for
the A.rhizogenes mediated transformation. Transformation with A.rhizogenes was
carried out with the plants growing in green house through their in-vitro propagation.
Hairy roots were produced from A. rhizogenes strain LBA9402 infected stem portions
of A. annua and A.dubia after seven days of infection but no hairy roots were
produced from strain LBA8196. Transformed and control roots were cultured on solid
B5 medium for further roots proliferation. Transformed roots showed better
proliferation than control roots.
Artemisinin content was significantly increased in transformed material of both
Artemisia species when compared to un-transformed plants. The artemisinin content
increased mostly five to ten times within leaves of transformed lines, hairy roots and
roots of transformed shoots. It indicated the plant capability of synthesizing much
higher amounts than has been achieved so far through traditional breeding. Similarly,
amount
of
different
derivatives
of
artemisinin
i.e.
artemether,
arteether,
dihydroartemisinin and artesunate was also significantly increased in transformed
material of both Artemisia species when compared to un-transformed plants.
Expression of all the tested genes involved in artemisinin biosynthesis
pathway was significantly increased, although variation amongst the genes was
observed. Cytochrome P450 (CYP71AV1) and aldehyde dehydrogenase 1 (ALDH1)
expression levels were higher than that of amorpha-4, 11 diene synthase (ADS).
Levels of the trichome development and sesquiterpenoid biosynthetic gene (TFAR1)
expression were also found increased in all transgenic lines. Trichome density
significantly increased in the leaves of transformed plants, but no trichomes were seen
in control or transformed roots. Crude hexane and aqueous extracts of rol genes
transformed plants revealed higher anticancerous activities against MCF-7 breast
cancer cell lines. Hexane extracts of transgenic plants revealed higher anticancerous
activity against MCF-7 breast cancer cell lines compared to aqueous extracts.
Transcriptomic study of A.annua and A.dubia allowed sequencing of the
transcriptome of these species for the first time. 16400 Contigs were generated by
aligning different transcriptome sequences. Up and down regulation and Putative gene
functions were predicted. BLAST of 500 contigs was performed, out of which 264
contigs showed homology with genomes of different organisms. Blast results of some
contigs showed that some species have genes that are similar to those involved in
artemisinin biosynthesis pathway. It would be interesting to know what are the
pathways in which those genes are involved in these species. Furthermore, divergence
of A. annua and A.dubia from the common ancestors can be found through
phylogenetic tree construction.
Chapter No. 1
Introduction and Review of Literature
INTRODUCTION AND REVIEW OF LITERATURE
Malaria is a disease that causes over one million deaths each year, putting 40%
of the world population at risk (WHO, 2004) (Fig 1.1). There are four parasites that
cause malaria but the one that causes the most illness and death is Plasmodium
falciparum. This is because some strains have become resistant to many of the
affordable current treatments including, chloroquine, quinine, mefloquine, and
primaquine (Balint, 2001).
Fig 1.1: Malaria Distribution and Statistics (WHO, 2004)
In 1972, China was the first country which purified an important antimalarial
drug and discovered its molecular structure. It was named qinghaosu, a sesquiterpene
from the plant Artemisia annua L. today it is known as artemisinin (Meshnick et al,
1996). Artemisinin and its derivatives have been found effective against all the stages
of resistant strains of P. falciparum (Balint, 2001).
Although artemisinin has been found as very useful medicine yet its production
is very low as compared to its required amount. The World Health Organization,
WHO, estimated that 130 million treatments would be needed in 2010, requiring 330
tons of artemisinin (WHO, 2008). This presents a problem because natural plant
produces very low amount of artemisinin. Only 6 kg of artemisinin is produced from
one ton of dry A. annua leaves. Researchers are trying to produce an increased
amount of artemisinin in different species of Artemisia plants because this drug also
cannot be produced economically through organic synthesis (Abdin et al., 2003).
1.1: Genus Artemisia, a natural source of artemisinin
Artemisia (Qinghao) is one of the most ancient plants commonly known as
―worm wood‖ that has persistently remained a traditional Chinese herb. It has been
described in China as having medicinal properties since 168 BC (Anon, 1982). Ge
Hong (281–340 B.C.), in his book ―Handbook of Prescriptions for Emergency
Treatment‖, recommended tea-brewed leaves to treat fever and chills. Afterward,
antimalarial activity of Artemisia was described in 1596 by Li Shizhen in his book
―Ben Cao Gang Mu‖ (Compendium of Materia Medica) (Efferth, 2009).
An antimalarial research program was started by Chinese Government during
Vietnam War to search for antimalarial secondary metabolite systematically. As a
result, artemisinin (qinghaosu), an active antimalarial secondary metabolite, was
isolated in 1972 from A. annua L (Anon, 1981). In 1979, structure of this antimalarial
principal was determined (Liu et al., 1979). Artemisinin was a sesquiterpene lactone
of terpenes family containing an endoperoxide bridge, named artemisinin or
Qinghaosu (Chinese) according to scientific classification of natural products.
1.1.1 Evidence of presence of artemisinin in some Artemisia species other than A.
annua
Presence of artemisinin in cosmopolitan species, A. annua has been reported
in a large number of publications around the globe (Woerdenbag et al., 1990; 1994;
Wallaart et al., 2000; Kumar et al., 2002). Although presence of artemisinin in all
Artemisia species was suggested by Moore, (1979) yet he had no concrete evidence to
support his claim however, further progress in research proved the presence of
artemisinin in other species of Artemisia. According to one report artemisinin is
present in A. apiacea and A. lancea but only in minor quantities (Hsu, 2006). Zia et
al. (2007) described the presence of artemisinin in leaves of A. absinthium. Arab et al.
(2006) reported the presence of artemisinin in aerial parts of A. sieberi. Another report
also showed the presence of artemisinin in A. dubia and A. indica (Mannan et al.,
2008). A. cina is another discovered source of artemisinin (Aryanti et al., 2001).
Several naturally growing Artemisia species in Pakistan also contain artemisinin in
their various parts (Mannan and Mirza, 2004).
1.1.2: Evidence of antimalarial activity of some Artemisia species other than A.
annua
There are several reports of antimalarial activity of various species of
Artemisia supporting the claims of artemisinin presence in species other than A.annua
as well. Zafar et al. (1990) reported antimalarial activity of A. absinthium. Yarnell and
Abascal, (2004a; 2004b) also showed the presence of antimalarial activity in extract
of A. vulgaris. Valecha et al., (1994) claimed significant antimalarial activity of
ethanol and petroleum extracts of A. japonica, A. maritima, and A. nilegaric in rats.
Likewise, methanolic extract of A. persica also showed antiplasmodial activity
(Sadeghpour et al., 2006).
1.1.3: Taxonomic classification of genus Artemisia
The taxonomic classification of genus Artemisia according to Mabberley,
(1997) is as follows:
Class: Magnoliopsida
Subclass: Asteridae
Order: Asterales
Family: Asteraceae
Genus: Artemisia
Artemisia is one of the largest genera in the family Asteraceae (Martin et
al., 2001; 2003; Watson, 2002) and a well-known cosmopolitan wind pollinated genus
as well. There are many publications related to its phylogenetic relationships,
including its molecular phylogeny (Heywood and Humphries, 1977; Ling, 1982;
Watson, 2002; Valles et al., 2003) and distribution (Ling, 1988; 1992; 1994a; 1995;
Valles et al., 2003). However, detailed study related to its origin and subsequent
development in the geological past, is still lacking.
Linnaeus first described this genus as containing 19 species in his Species
Plantarum (Linnaeus, 1753). In his later work, most of the species were given their names
according to their origin e.g. species reported from Ethiopia was named A. aethiopica L.
and that from china as A. chinensis L. Afterward, Hooker and Thomson, (1881) further
classified it into sections and placed the species in it according to their annual, biennial or
perennial nature. Clapham et al. (1962) recognized seven species through morphological
characters, and grouped them into sections. Cullen, (1975) described Artemisia consisting
of twenty-three species. There, he focused upon leaf morphology for the identification of
species.
Later scientists placed Artemisia into various groups like Podlech, (1986) and
another group of scientists placed Artemisia in six to eight genera within its taxonomic
boundaries. Moreover Artemisia of antiquity was divided into three genera Artemisia,
Absinthium and Abrotanum (Cronquist, 1955; 1988; Bremer and Humphries, 1993; Ling,
1994b; Kornkven et al., 1998; 1999; Torrel et al., 1999; Martin et al., 2001).
Many local and common names are now used all over the world for a number of
eminent Artemisia species. These are Annual wormwood (A. annua), Grand wormwood
(A. absinthium), Tarragon (A. dracunculus), Russian Wormwood (A. vestita or A. gmelin)
and Mugwort (A. vulgaris).
1.1.4: History of Artemisia
Artemisia is a large, assorted group of plants with between 200-400 species
belonging to the daisy family Asteraceae and it is well known since ages through the
study of taxonomy. It comprises hardy herbs and shrubs known for their volatile oils
(Watson, 2002). According to Cullen‘s (1975) view, Artemisia is a late flowering and
well pollinated genus.
1.1.5: Morphology
Artemisia is annual, biennial or perennial, usually bitter-aromatic or nonaromatic glabrous, hairy or punctuate-glandulase herb or small to large shrub with tap
root or horizontal root stock. Leaves alternate, basal large and petiolate, upper often
subsessile to sessile, undivided or toothed-shallowly to deeply incised or lobed,
palmati or pinnatipartite or 2-4 pinnatisect.
Synflorescence a pancicle, sometime racemoid or spicate, occasionally globoid
or fascieled capitulla generally numerous, small, rarely mediocre, nodding or erect,
oblong-cylindrical or ovate to almost globose, hetorogamacy. Involucre ovoid to
campanulate or hemispherical, phyllaries in 2-4 (-6)-sereis imbricate, hairy (or)
glabrous inner one mostly scarious-margined or scarious. Receptable plane, convex,
conical or hemi-spherical, glabrous or pilose (Fig. 1.2a and 1.2b).
Flowers brownish, reddish or yellowish, all tubular. Ray-florets: pistilate and
fertile, corolla narrowly tubular, gewrally tapering upwards, 2-3(-4) toothed 1 ±
oblique, eligulate, style exerted, 2-cleft, branches recurved, linear filiform and tereteoblong, ± flattened. Discflorets bisexual, fertile or sterile, corolla tubular to ±
companulate or funnel shaped, 5 toothed, anthers oblong, larger than the filaments,
obtuse or ± cordate at the base, apical appendages lanceolate or subulate, style
exserted or included, either 2-cleft with flat, truncate and apically erose or fimbriate, ±
recurved branches or columnar with an erose or fimbriate disc or cup at the apex.
Cypselas of both marginal and disc (when fertile) terete, oblong-ellipsoid or obvoid to
nearly fusiform-prismatic, faintly many striate or smooth, glabrouse, apecially
rounded or trunate. Pappus absent or sometimes cypselas with a minute annulus or
scarious corolliform ring (Ghafoor, 2002)
Figure 1.2: Floral morphology of Artemisia (A) A daisy flower (Asteraceae) – a
composite head of many small flowers (B) Detailed description of floral parts.
1.1.6: Distribution
Artemisia species are broadly distributed in north temperate regions of both
new and old world, extreme west and South Africa, South America (Ghafoor, 2002),
Asia, west America, Canada, New Zealand (Stewart, 1972), Afghanistan, and extends
westwards to the Atlantic (Said, 1969).
Artemisia dubia L. grows wild in northern India, China, Xizang, Nepal and
northern hilly areas of Pakistan specially Swat, Parachinar, Abbottabad, Hazara, Bagh
(Haq, 1983), Chitral, Srinagar, Pahlgam, Tsrar Sharif, Skardu, Ladakh, Kurram
valley, Kishtwarpass (Ghafoor, 2002). Artemisia is highly rich in many areas of
Kashmir between altitudes of 5000-7000 ft. (Basu and Kirtikar, 1988; Dasture, 1952).
It has also been reported in Margalla hills of Islamabad (Rizwana et al., 2002).
Artemisia annua L. is inhabitant to Asia, most probably to China (McVaugh,
1984). A. annua occurs as a part of steppe vegetation in China at 1000 to 1500 meter
above sea level (Wang, 1961). It is also commonly found as a wild weed and as a
roadside plant. Plant now grows wild in many countries, such as Argentina, Bulgaria,
France, Hungry, and Romania and is cultivated for its essential oil in Italy, Spain,
Eastern Canada, United States and former Yugoslavia (Said, 1969; Klayman, 1989;
1993).
According to Rizwana et al., (2002) 30 species of Artemisia are growing
naturally in Pakistan. Another publication indicates the presence of 32 species
(Stewart, 1972). However ten-species found in Pakistan are also available in Japan,
China and Russia (Junshen et al., 1996). Artemisia is also found in Saudi Arabia
(Ghafoor and Al-Turki, 2000). Hajra illustrated 33 species of Artemisia from India
(Hajra et al., 1995; Dasture, 1952; Palumin and Stainton, 1992).
1.2: Morphology of Artemisia annua
Artemisia annua is an annual herb inhabitant in Asia, especially in China. The
name of the plant is qinghao. Argentina, Bulgaria, France, Hungary, Italy, Romania,
Spain and many other countries all over the world has naturalized Artemisia annua
(Ferreira et al., 1996).
A.annua is an annual, biennial, or perennial weed reaching about 2 m in height
with alternate branches. Leaves are deeply dissected, with an aromatic odour, 2.5 to 5
cm in length, 1 to 3 cm in width. Flowers are tiny and yellow; in lose panicles with
capitula 2 to 3 mm across. There are central and marginal florets. The seed vessels
consist of one achaene, faintly nerved and 1 mm long. Naturally the plant is pollinated
by insects and by the wind (Hansel et al., 1992) (Fig 1.3).
Figure 1.3: Artemisia annua
Artemisia annua is annual, biennial, or perennial, usually bitter aromatic or nonaromatic, glabrous, hairy or punctate-glandulose herb or small to large shrubs with
taproot or horizontal rootstock. Leaves alternate, basal large and petiolate, upper often
sub sessile to sessile, undivided or toothed-shallowly to deeply incised or lobed,
palmate- or pinnatipartite or 2-4 pinnatisect. Synflorescence a panicle, sometime
racemoid or spicate, occasionally globoid or fascicled. Capitula generally numerous,
small, rarely mediocre, nodding or erect, oblong-cylindrical or ovate to almost
globose, heterogamous. Involucre ovoid to campanulate or hemispherical: phyllaries
in 2-4 (-6)-series, imbricate, hairy or glabrous, inner ones mostly serious-margined or
scarious. Receptacle plane, convex, conical or hemispherical, glabrous or pilose.
Flowers are brownish, reddish or yellowish, all tubular. Ray-florets: pistilate and
fertile; corolla narrowly tubular, generally tapering upward, 2-3(-4)-toothed, ±
oblique, eligulate; style exerted, 2-cleft, branches recurved, linear-filiform and tereteoblonge, ± flattened. Disc-florets bisexual, fertile or sterile; corolla tubular to ±
campanulate or funnel-shaped, 5-toothed; anthers oblong, longer than the filament,
obtuse or ± cordate at the base, apical appendages lanceolate or subulate; style
exserted or included, either 2-cleft with flat, truncate and apically erose or fimbriate, ±
recurved branches or columnar with an erose or fimbrate disc or cup at the apex.
Cypselas of both marginal and discs (when fertile) terete, oblong-ellipsoid or
obovoid to nearly fusiform-prismatic, faintly many straite or smooth, glabrous,
apically round or truncate. Pappus absent or sometimes cypselas with a minute
annulus or scarious corolliform ring (Ghafoor, 2002).
1.3: Morphology of Artemisia dubia
Artemisia dubia is a peremial herb with several erect, 1-1.8m tall, sulcate,
purpureus glabrescent, staut stems from woody, upright root stock as shown in figure
1.1. Leaves shortly petiolate to almost sessile, oblong-elliptic to broadly ovate, 8-12 x
6-9 cm, green and white dotted above along with or without sparse T-shaped hairs,
grayish-green arachnoiad hairy beneath to almost glabrous, bipinnatisect, primary
segments, elliptic-lanceolate, 3-4.5 x 1.5-2.5 cm, acute mueronate, secondary
segments ± elliptic lanceolate, 1-1.5 x 2.5-4.5 mm, acute or ± obtuse revolute;
uppermost leaves linear lanceolate, with or without basal auricles. Capitula numerous,
heterogamous, oblong-companulate, 3-3.5 x C.2 mm, ± approximate, almost sessile,
in a narrow to broad, 15-30 x 10-20 cm panicle with ascending to ± patent (upper),
upto 25 x 2-3 cm primary braneles (Ghafoor, 2002). Involcure 4-seriate, phyllaries
loosely imbricate, all sparsely arachnoid hairy outside, outermost narrowly ovate,
green, acute, median elliptic to narrowly obovate, 2.5 x 1 mm, obtuse, widely scarious
hyaline on margins and apex. Receptacle ± flat, glabrous (Fig 1.4).
Figure 1.4: Artemisia dubia
Involcure 4-seriate, phyllaries loosely imbricate, all sparsely arachnoid hairy
outside, outermost narrowly ovate, green, acute, median elliptic to narrowly obovate,
2.5 x 1 mm, obtuse, widely scarious hyaline on margins and apex. Receptacle ± flat,
glabrous. Florets 16-20 purplish, all fertile; marginal florets 6-8 with C. 1mm large,
basally broadened, bidentate, glandulose corolla tube and long exerted style branches,
disc florets 10-12, with C.1.75mm long, 5 toothed, glandulose apically very sparsely
hairy corolla tube. Cypselas brown, 1.25-15 mm large, with terminal carolliform scar
(Ghafoor, 2002).
Artemisia dubia is a common weed in open localities, fallovo fields, waste
places, and roadsides, rare in regularly cultivated fields. Propagate mainly by
underground stolens. inhabitant of Europe, cartimental Asia, China, Pakistan,
Northern India, and Nepal, used as a medicine in various ways. Host plant for meloid,
gyne root knot nematodes. Blooming period is August to October (Ghafoor and AlTyrki, 2002).
1.3.1: Selection of Artemisia dubia for artemisinin enhancement
Although artemisinin concentration is highest in Artemisia annua, there are
reports describing the presence of artemisinin in Artemisia absinthium, Artemisia
dubia and Artemisia indica (Arab et al., 2006; Zia et al., 2007; Mannan et al., 2008).
Chemical synthesis of artemisinin is difficult and therefore other sources such as other
Artemisia species or tissues such as plant callus (He et al., 1983), shoot (Fulzele et al.,
1991) and hairy root cultures (Qin et al., 1994) offer attractive alternatives which
could be grown throughout the year distancing production from time of harvest.
Mannan et al. (2008) through Agrobacterium rhizogenes transformation increased the
artemisinin content within hairy roots of Artemisia dubia and Artemisia indica to
0.6% and to 0.23% respectively. Furthermore, A.annua is restricted to certain global
regions due to its growth requirements. A.dubia is capable of growth in more varied
conditions and could offer an alternative for production ensuring security of
production which would help ensure a constant price.
1.4: Secondary metabolites and medicinal constituents of Artemisia
Artemisia is famous due to its distinguished therapeutically significant
constituents and has undergone extensive phytochemical investigations over the past
two decades. A large number of sesquiterpines have been isolated (Sy and Brown,
1998; 2001) until now from this genus. Most important one is artemisinin (Tu et al.,
1982). Artemether, arteether and artesuate are most widely used derivatives of
artemisinin which are the methyl ether, ethyl ether and hemisuccinate ester of
dihydroartemisinin respectively (Anon, 1982; Li et al., 1982). Artemisinin derivatives
have been studied for their efficacy as antimalarial agent.
Constituents which have been isolated from its volatile oils (0.5 to 1.6% oil/fresh
weight) include α and β thujone (0.042%), phellandrine (12.3%), thygylalcohal
(4.5%), azulene (2%), glycoside (0.003%), resin (5.2%), starch (1.45%), alpha-pinene
(0.032%), camphene (0.047%), β-pinene (0.882%), myrecene (3.8%),1,8-cineole
(5.5%), Artemisia ketone (66.7%), linalool (3.4%), camphor (0.6%), barneole (0.2%),
and β-caryophylene (1.2%) (Srivastava, 1999).
Other constituents isolated include flavonoids (Hoffmann and Hermann, 1982),
ascorbic acid (Slepetys, 1975), carotenoids (Sergeeva and Zakharova, 1977), Tannins
(Slepety, 1975), lignans (Greger and Hofer, 1980), Pinitol, artemin, ridentin,
santolineal, stigmasterol, daucosteral, sesanin, beta steral, alpha amyrin, Judaicin
(cardio tanic), cirsimaritin and glaucolide like sesquiterpine lactones (Tan et al., 1999;
Khafagy and Tosson, 1968; Khafagy et al., 1988; Galal et al., 1974). Artemisia plants
still represent a large number of unexplored structurally novel compounds that might
act as lead for the development of new drugs (Hostettmam, 1987).
1.5: Medicinal importance of genus Artemisia
Phytopharmacological evaluation of Artemisia species shows the presence of
anti-inflammatory (Sommer et al.,1965), antipyretic (Ikram et al.,1987), antifertility
(Rao et al., 1988), antibacterial (Kaul et al.,1976), antifungal (Maruzzella et al.,
1960), anthelmintic (Caius and Mahasber, 1920), antiamoebic (Tahir et al., 1991),
antimalarial (Hernandez et al., 1990; Zafar et al., 1990), antihepatitic (Gellani and
Jambaz, 1995), hepatoprotective (Kiso et al., 1984; Handa et al., 1986; Oshima et al.,
1984) and antidiabetic activities (Tan et al., 1999).
Studies show that some species of Artemisia (A. judacia L.) have cardiotonic
effect (due to Jundaicin) that does not deviate from the general frame work of
dagitoxin action (Galal et al., 1974). Antiviral and antibacterial activities are due to
the cirsimaritin, which also has inhibitory effect on several mammalian enzymes
(Abdalla and Abu-Zarga, 1987). There may be synergism of components too as
shown by methoxylated flavenoids of Artemisia dubia, which enhance the activity of
artemisinin (Elford et al., 1987).
Artemisia annua leaves as well as flowering tops are used medicinally but
leaves are preferred. The fresh plant is always more effective than the dry plant. The
green tops of this herb are excellent remedy for disorders of the stomach (Dasture,
1952). Traditionally it is used in different cultures as insecticide (Said, 1996) and
vermifuge (Caius, 1986). The whole plant is an aromatic tonic and formerly enjoys a
high reputation in debility of the digestive organs (Caius, 1986; Basu and Kirtikar,
1988).
This species also act as anemma gogue in amenorrhoea, caused by uterine
disorders or general debility. It is also prescribed in nocturnal pollutions, chlorosis,
anaemia, wasting disease (Dasture, 1952), headaches, migraine, paralysis, facial
paralysis, spasmodic affections (such as epiplepsy), and hysteria in nervous irritability
nervous disorders and piles. It is an effect diuretic also causes perspiration (Said,
1996). A week decoction of the plant is given to children in measles (Dasture, 1952).
Many of its constitutents are used as flavouring agents (Lee and Geismam, 1970;
Marco and Barbera, 1990; Heinrich et al., 1998; Sy and Brown, 2001).
It is considered effective for jaundice and dropsy. It is used as a tonic either alone
or in combinations in atonic diseases of the digestive system (Dasture, 1952). Infusion
of top of fresh plants has excellent effect for all disorders of the stomach, creating
appetite, promoting digestion and preventing sickness after meals (Caius, 1996). In
suitable combinations it is applied as paste over the corresponding area of the
abdomen to reduce liver and spleen inflammations (Said, 1969). The herb is
commonly prescribed in the form of a poultice or fermentation as an antiseptic and
discutiend (Caisus, 1986; Basu and Kirtikar, 1988). Wood tea, or powdered herb in
small doses mixed in a little soup is used to relieve bilians melancholia. It is also used
in intermittent fever (Caius and Mahaskar, 1920).
Indigenous system of medicine uses its leaves and flowering tops in crude
form as anthelmintic, antiseptic, febrifuge and stomachic. It has been employed
successfully to alleviate chronic fever, dyspepsia and hepatobilary ailments (Said,
1982). Artemisia plant has great potential, much work need to uncover its other
activities. That is why; this plant is called ―the plant of future‖.
1.5.1: Non medicinal importance of the genus Artemisia
Various constituents obtained from A. pallens (Srivastava, 1999) and A. annua
(Charles et al., 1991) have very good fragrance, which can be used in manufacture of
high-grade perfumes. A number of specific essential oils obtained from A. annua
(Anon, 1992) and A. absinthium (Arino et al., 1999) have wonderful taste that are
being used for the flavoring of vermouth and in food industry, respectively.
1.5.2: Toxic effects of Artemisia
Numerous species of Artemisia are unhealthy. Periodic uses and large dose of
Artemisia causes restlessness, vomiting, vertigo, tremors and canvulsions (Said,
1969). Its affinity to produce headaches and other nervous disorders is well known by
travelers in Kashmir and Ladak (Caius, 1986; Basu and Kirtikar, 1988). The juice of
the large leaves of Artemisia species is highly nauseous (Dasture, 1952). Many
Artemisia species are sources of allergies in humans (Lewis, et al., 1983). Large doses
of volatile oils are effective narcotic poison (Caius, 1986).
1.6: Artemisinin
Artemisinin an endoperoxide sesquiterpene lactone produced by aerial parts
of Artemisia annua L. is effective even against multi-drug resistant strains of the
malarial parasite. The isolation and characterisation of artemisinin from Artemisia
annua is considered as one of the most novel discoveries in recent medicinal plant
research. It was isolated from the plant in 1972 (Roth and Acton, 1989) and in 1979,
its structure was determined by X-ray analysis (Brown, 1993). It has an empirical
formula of C, H, O. Artemisinin has a peroxide bridge to which its antimalarial
properties are attributed. It has a unique structure and lacks nitrogen containing
heterocyclic ring, which is found in most anti-malarial compounds as shown in figure
1.4. Artemisinin is an odourless, colourless compound and forms crystals with
melting point of 156 - 157 O C. The molecular weight as determined by high
resolution mass spectroscopy is m/e 282.1742 m+ (Brown, 1993) (Fig. 1.5).
Fig.1.5: Structure of Artemisinin, C15O5H22 (MW=282)
Artemisinin has proved to be one of the most promising drugs. It has also
shown to possess considerable antimicrobial and antifungal activities (Dhingra et al.,
2000). Although the complete organic synthesis has been established, but chemical
synthesis of artemisinin is not yet economically feasible because of its complexity and
low yield. Currently the leaves, roots and flowers of Artemisia species form the only
source of this drug. Artemisinin is found in very low quantities (0.05%-1.1%) in
different cultivars of Artemisia dubia and Artemisia annua (1%-4%). High
artemisinin yielding clones are being isolated by selection and other non-conventional
approaches; however, these have their own limitations. Therefore, in the recent past,
in vitro culture system of A.annua has been exploited for the production of
artemisinin (Delabays et al., 1993).
1.6.1: Artemisinin synthesis and storage
Reports on the distribution of artemisinin throughout the plant are inconsistent. In
some studies Artemisinin has been reported to be higher at the top of the plant
(Charles et al. 1990; Laughlin 1995) while others suggest it is equally distributed
(Laughlin 1995). Artemisinin has also been shown to be produced by differentiated
(shoots + roots) shoot cultures (Martinez and Staba 1988; Fulzele et al., 1991;
Whipkey et al., 1992; Ferreira and Janick 1996b) but in shoots without roots only
trace levels are found suggesting a regulatory effect (Martinez and Staba 1988; Jha et
al., 1988; Fulzele et al., 1991; Woerdenbag et al., 1993; Paniego and Giuliette 1994).
Most workers (Martinez and Staba 1988; Tawfiq et al., 1989; Fulzele et al., 1991;
Kim et al., 1992) were unable to detect artemisinin in roots, although Nair et al.,
(1986) and Jha et al., (1988) reported trace amounts.
Detection of artemisinin from seeds appears to be due to the presence of floral
debris (Ferriera et al., 1995b). The highest concentration of artemisinin is found in the
inflorescence, which may contain more than ten times as much artemisinin as leaves
(Ferriera, et al., 1996).
The current understanding is that artemisinin is produced in 10-celled glandular
trichomes located on leaves, floral buds, and flowers (Ferreira et al., 1995; Tellez et
al., 1999; Olsson et al., 2009) and sequestered in the epicuticular sac at the apex of
the trichome (Olsson et al., 2009). For instance, artemisinin concentrations are higher
in leaves that are formed later in development than in leaves formed early in the
plant‘s development; this difference has been attributed to a higher trichome density
and a higher capacity per trichome in the upper leaves (Lommen et al., 2006).
Artemisinin biosynthesis was thought not to be synthesized in roots (Ferreira et al.,
1995) or pollens. ELISA analysis has shown that green roots accumulate artemisinin
(0.001% dry weight) which was confirmed by GC-MS analysis (Jaziri et al., 1995). It
has been shown that hairy roots produced by infection with Agrobacterium rhizogenes
can produce artemisinin (Jaziri et al., 1995; Liu et al., 1999). Weathers et al., (1994)
reported levels of artemisinin (0.4%) comparable to that found in the leaves within
hairy root cultures of A. annua transformed with Agrobacterium rhizogenes. This
would suggest that the plant is capable of producing artemisinin in the absence of
trichomes. However, to date, the regulatory mechanisms that control artemisinin
biosynthesis and its formation outside the trichomes are poorly characterized.
1.6.2: Derivatives of artemisinin
Derivatives of artemisinin like ether derivatives artemether and arteether, its
primary metabolite artenimol and the ester derivative artesunate (Fig 1.6) are
exhibiting superior activity against Plasmodium falciparum and also against
Plasmodium vivax. The reactivity of endoperoxide bridge which is the common
structural feature of artemisinin and all its derivatives is the key for their biological activity.
Although artemisinin derivatives are fast acting substances which can rapidly
remove the malarial parasites from blood but due to its short biological half-life which
preclude its long lasting activity making artemisinin derivatives less preferable in
monotherapy, but these derivatives can be used in combination with longer-acting
drugs that have a slower onset of activity (Davis et al., 2005).
Artemisinin (Qinghaosu)
Artenimol (Dihydroartemisinin)
Artemether
Artesunate
Arteether
Fig. 1.6: Chemical structure of artemisinin and its derivatives
The WHO Roll Back Malaria programme has advocated such a strategy,
recommending the use of artemisinin-containing therapies (ACT) in areas of
emerging, high resistance to the most commonly used antimalarial (WHO, 2004).
Also, a recent publication has reviewed the use of artemisinin-based combination
therapies in uncomplicated malaria (Davis et al., 2005). Another asset of artemisinin
is their apparently excellent human safety and tolerability (Price et al., 1999).
1.6.3: Mode of action of artemisinin
The only one group of compounds for which malarial parasites have not yet
developed the resistance are the artemisinin and its derivatives, increasingly being
used. It is effective even against chloroquine-resistant and chloroquine sensitive
strains of Plasmodium falciparum as well as against cerebral malaria (Anon, 1979; Li
et al., 1982; Roth and Acton, 1989; Pras et al., 1991). The unusual structure of
artemisinin is the basis of its potent antimalarial activity, which makes it distinctive in
mode of action from other antimalarial drugs. There is an assumption that interaction
of artemisinin with heme leads to death of the parasite because malarial parasite
contains large amount of heme iron (Meshnich et al., 1993; Kamchonwongpaisan and
Meshnich, 1996; Meshnich, 1996). Yet, another experiment (Eckstein et al., 2003)
showed that artemisinin is activated by ferrous iron inside the parasite, which is
independent of heme, the source of this iron is not known.
According to Kamchonwongpaisan and Meshnick, (1996) mode of action of
artemisinin completes in two steps. First step involves the production of oxygen free
radical by the cleavage of the endoperoxide linkage of artemisinin by the heme iron;
therefore this step is called as activation step. Oxygen free radical is subsequently
rearranged to give a carbon free radical. The second step involves an alkylation of
specific malarial proteins by the carbon free radical, which causes a lethal damage to
malarial parasite; therefore this step is called an alkylation step (Fig 1.7).
Figure 1.7: Mechanism of the reductive activation of artemisinin by iron (II)heme, leading to covalent heme-artemisinin adducts.
1.6.4: Biological activities of artemisinin
Artemisinin is not only effective against malaria, but it has also been
demonstrated to be effective against a range of other diseases, such as schistosomiasis
(Borrmann et al., 2001), hepatitis B (Romero et al., 2005), and a variety of cancer cell
lines including breast cancer, human leukemia, colon, and small-cell lung carcinomas
(Efferth et al., 2001; Singh and Lai, 2001). Artemisinin is comparatively a safe drug
and can be used even for pregnant women without any adverse side effects (Dellicour
et al., 2007). With other anti-microbial activities it is also effective against drug
resistant cancers (Efferth et al., 2002; Sadava et al., 2002).
Artemisinin also act as a natural herbicide because it has been found to be an
effective plant inhibitor which contributes to its allelopathic activity (Duke et al.,
1987; Chen et al., 1991; Stiles et al., 2005). Entire biological activities of artemisinin
are represented in figure 1.8.
Figure 1.8: Diagramic representation of biological activities of artemisinin.
1.6.4.1: Malaria
A serious disease which is the basis of at least 1 million deaths of children
under 5 year, and affected more than 247 million people worldwide and due to which
3.3 billion people were at the risk in 2006 is the disease malaria. According to one
report of World health organization (WHO, 2008a), 0.0014 million people were died
in 2006 due to 1.5 million malarial successions about which 30% cases were due to
the Plasmodium falciparum and one hundred nine countries were affected with
malaria in 2008. Malarial parasites which infect human beings are Plasmodium
falciparum, Plasmodium vivax, Plasmodium malariae and Plasmodium ovale.
Eradication of this disease is becoming very difficult every day due to the
development of resistance against multi-drugs system and pesticides by Plasmodium
species and the infection vector, the Anopheles mosquito (Winstanley et al., 2002). A
large number of publications around the world came in print on the impact of malaria
on economic and social development because malaria has a terrific impact on both
economic and social development. Shortly, one can agree with the statement that
―where malaria prospers most, human societies have prospered least” (Sachs and
Malaney, 2002).
1.6.4.2: Schistosomiasis
After malaria, the second most socioeconomically desecrate waterborne
disease is bilharziasis, which is commonly known as a schistosomiasis, found in much
of the Third World (Warren, 1987). As concern to the socioeconomic and public
health, The World Health Organization ranks schistosomiasis as one of the major
tropical diseases (WHO, 2008b). Five species Schistosoma mansoni, Schistosoma
intercalatum, Schistosoma haematobium, Schistosoma japonicum and Schistosoma
mekongi which infect human beings, effects as many as 600 million people as they
perform their daily activities related to water such as swimming, fishing, farming,
washing, and bathing. In its extremity, schistosomes can survive for decades in their
human host, while schistosomiasis is predominantly a disease of the rural poor;
travellers to endemic areas are also subject to infection. Although artemisinin is very
cogent against this disease but its mode of action is not clear (Garfield, 1986).
1.6.4.3: Hepatitis B
All over the world about 2 billion people have been infected with virus and
others 350 million people live with incessant infection due to a disease hepatitis B that
is 100 times more dangerous than AIDS. According to World Health Organization
(WHO, 2008c), every year 0.6 million people die due to acute or chronic effects of
hepatitis B, however it is less common in Pakistan (Andre, 2000). Hepatitis B is a
viral infection that damages the liver and it can be of two types, acute infections run a
course of several months, and chronic infections are often lifelong which lead to liver
failure with cirrhosis and hepatocellular carcinoma (Seeger and Mason, 2000).
Hepatitis B virus is DNA virus of unique class (Robinson et al., 1974). It is
transmitted between people by contact with the blood or other body fluids (i.e. semen
and vaginal fluid) of an infected person. Although artemisinin therapy is very
effective against this disease but its precise mode of action is unknown (Peterson et
al., 1976; WHO, 2008c).
1.6.4.4: Cancer
Cancer is a foremost cause of death all over the world. According to different
data published all over the world it is described that cancer caused 7.9 million deaths
(around 13 % of all deaths) in 2007 and this number is increasing every day with an
estimation of 12 million deaths in 2030. The different types of cancer which cause
maximum number of deaths every year include Lung, stomach, liver, colon and breast
cancer. Cancer cells are produced due to changes in cell acquired by external agents
or by inherited genetic factors. According to World Health Organization deaths due to
cancer can be prevented up to 30% (WHO, 2008d).
To date, the most considerable pharmacological studies on anticancer use of
artemisinin have been carried out which explain mechanism of action of artemisinin
against cancer in very effective way (Efferth et al., 2001; Singh and Lai, 2001; Efferth
et al., 2002; Sadava et al., 2002). According to these considerations, cancer cells
require large amount of iron for their multiplication and to express a large
concentration of cell surface transferrin receptors that facilitate uptake of the plasma
iron-carrying protein transferrin via endocytosis, artemisinin reacts with iron to form
free radicals which can kill cells. By covalently tagging artemisinin to transferrin,
artemisinin could be selectively picked up and concentrated by cancer cells. In
addition, both artemisinin and iron would be transported into the cell in one package.
Once an artemisinin-tagged transferrin molecule is endocytosed, iron is released and
reacts with tagged artemisinin. As a result, free radicals form that kill the cancer cells.
Artemisinin-tagged transferrin is highly selective and potent in killing cancer cells.
Thus, artemisinin and artemisinin-tagged iron-carrying compounds could be
developed into potent anticancer drugs (Rowen, 2002; Henry et al., 2005).
1.6.4.4.1: Effect of artemisinin and its derivatives on different types of cancer
A large number of studies carried out by different laboratories all over the world
now explain the effect of artemisinin and its derivatives on different types of cancer,
such as breast cancer (Posner et al., 2004; Nam et al., 2007; Sing and Lai, 2004;
Thomas et al., 2003), lung cancer (Wu et al., 2006; Thomas et al., 2003), prostate
cancer (Chen et al., 2004; Thomas et al., 2003),), head and neck cancer (Yamachika
et al., 2004), bladder carcinomas, renal carcinoma (Thomas et al., 2003), ovarian
carcinoma (Jiao et al., 2007), cervical carcinoma (Disbrow et al., 2005), pancreas
carcinoma (Wu et al., 2001), colon carcinoma (Thomas et al., 2003), thyroid
medullary carcinoma (Yamachika et al., 2004), endometrial carcinoma and oral
squamous cell carcinoma (Yamachika et al., 2004).
The mechanisms of action for their antitumor activities are not fully understood,
but may include selective cytotoxicity of cancer cells (Lai and Sing, 1995; Sing and
Lai, 2001), induction of apoptosis (Sing and Lai, 2004; Dell‘Eva et al., 2004),
modulation of gene expression (Efferth et al., 2002; Wu et al., 2006), causation of cell
cycle arrest (Dell‘Eva et al., 2004; Wu et al., 2001; Li et al., 2001) and inhibition of
angiogenesis (Dell‘Eva et al., 2004; Wu et al., 2006; Chen et al, 2003; Chen et al.,
2004).
1.6.4.5: Herbicides
Herbicides are basically chemicals used generally in agriculture, industry, and
urban areas for weed control because herbicides inhibit or interrupt normal plant
growth and development. According to Chen et al., (1991) artemisinin obtained from
A. annua gave same level of inhibition as 2, 4-D and glyphosate in mung bean
phaseolus and therefore showed good herbicidal activity against various weeds. Many
publications have came into print signifying that artemisinin has potential to be used
as natural herbicide (Duke et al., 1987; Chen and Leather, 1990; Yun and Kil, 1992;
Lydon et al., 1997) but much effort is needed to find out its mode of action as
herbicide.
1.7: Improvement of artemisinin content
At present, artemisinin and its derivatives are used in combination therapies for
the treatment of malaria (Haynes, 2006), and for treatment of numerous cancers and
viral diseases (Efferth, 2007; Efferth et al., 2008). Scientists all over the world are
now trying at their best to find out the different ways to increase the production of this
precious secondary metabolite to fulfil its increasing demand. Conversely, the major
barrier in the commercialization of artemisinin is its low production (0.01-0.8 %) and
accumulation in naturally growing Artemisia species (Abdin et al., 2003). At the
moment, other resources for its increased production are under investigation, for
example the genetic modification of bacteria and yeast, but these experiments at the
end only give the artemisinin precursors which need further steps for artemisinin
production (Newman et al., 2006; Ro et al., 2006; Shiba et al., 2007; Zeng et al.,
2008). Genetic improvement of natural varieties has been attempted but the maximum
yield of artemisinin from this route reached so far is 2% (Graham et al., 2010). Due to
complexity and high cost in of chemical synthesis (Schmid and Hofheinz, 1983;
Haynes 2006; Tue Nguyen, 2011), the biosynthesis of artemisinin via the plant is still
the most cost effective. Therefore it is an extensive need to develop and understand
the normal biosynthetic pathway of artemisinin to enhance the artemisinin production
in Artemisia species and to yield high artemisinin producing transgenic plants and
hairy roots for continuous production of artemisinin to meet the increasing demand of
this important secondary metabolite to save lives of human beings from many lethal
diseases.
1.7.1: Selection of best cultivar
Artemisia is a perennial, well-established plant and often woody in nature.
Different cultivars of Artemisia are available like Artemisia lactiflora, Artemisia
vulgaris, Artemisia frigida, Artemisia absinthium, Artemisia dubia. The only known
source of the artemisinin, which is uneconomical to synthesize chemically, is the plant
Artemisia annua. The A. annua plant of the family Asteraceae, indigenous to South
East Asia, is an annual herb/shrub, which has become naturalized or is in cultivation
as a horticultural or medicinal plant in many parts of Asia, Africa, Europe, America
and Australia. The natural populations and genetic resources of A. annua from
different areas are known to demonstrate considerable variability in the accumulation
of artemisinin in the leaves and the capitula of the plant (Charles and Simon, 1990).
The foliage and inflorescence have identified and used genotypes of A.annua with
high artemisinin content for developing agrotechnologies suitable for cultivating this
industrial crop under a variety of soil and agroclimatic conditions (Woerdenbag et al.,
1994). Artemisinin yields ranging from 3.2 to 20 kg/h have been reported (Singh et
al., 1988; Woerdenbag et al., 1994).
1.7.2: Selection of different stages
In the vegetatively growing 20–30 weeks old plants of Artemisia, leaves are the
principle organs for the synthesis and accumulation of artemisinin; stems have
artemisinin in about 10-fold lower amount. Usually, the younger leaves have more
artemisinin than the older leaves. The leaves of young rosette plants during their
growth in the winter season (December through March) have very low concentrations
of artemisinin. The expression of artemisinin synthesis and accumulation in the leaves
progressively increases with the onset of summer in March/April and becomes high
by May/June (summer) and peaks during rainy season (July–September).The
flowering plants accumulate bulk of their total artemisinin in leaves (30%) and
capitula (40%). Since the presence of lipids (oil) in the achenes of capitula makes
artemisinin extraction cumbersome, the plants harvested in their vegetative state,
when > 90% of the artemisinin is in the leaves and fine stem, offer the best economy
in chemical extraction of artemisinin (Sushil et al., 2004; Mannan et al., 2010) Roots
lack artemisinin at all stages of plant growth, therefore tissue culture method is
adopted to increase the artemisinin content in roots.
1.7.3: Tissue culture of Artemisia
Several tissue culture studies have been carried out on various species of
Artemisia, findings of some of these are as follows.
In one study, round wormwood (Artemisia sphaerocephala Krasch) seeds were
germinated on MS medium without plant growth regulators. The hypocotyls of
seedlings were sliced and cultured on medium with 2, 4-D to induce callus (Xu and
Jia, 1996).
Much work has been done on Artemisia annua because of its antimalarial
activity. In this species callus can be obtained on media supplemented with
combinations of auxins and cytokinins, but non-friable callus is usually obtained.
Ferreira and Janick (1996) obtained the highest yield of friable callus with a
combination of BA at 4.44 M and 2, 4-D at 4.52 M. Vitamin C reduced browning,
and GA3 improved friability. However, after 17 months in culture, only 3 out of 24
clones kept as shoot cultures generated calli. Cell cultures were established using the
same callus-inducing medium, without agar. However, artemisinin was not detected
from callus or cell cultures or from the liquid medium.
Studies have been done to determine the effect of varying strength of B5
culture media, source and level of nitrogen in the media (nitrate and ammonium),
phosphate and the phytohormone, gibberellic acid (GA3), on both biomass and
artemisinin production in hairy root cultures. Preliminary results show that a 33%
increase in B5 medium, as increase in nitrate, an elimination of ammonia, and
addition of GA3 increase biomass yields. Artemisinin production was stimulated by
regular strength B5 (with lower levels of nitrate than for maximum biomass), low
levels of phosphate, and GA3 (Weathers et al., 1996).
In another study optimum proliferation of French tarragon (Artemisia
dracumulus L. var. sativa) short tips was obtained on MS medium supplemented with
1.8M NAA and 3% sucrose. After 4 weeks of culture, Maximum proliferation was
obtained with unpinched shoot tips placed horizontally on the medium. Maximum
rooting was observed with cutting>10mm in length. A 5-sec dip of the basal portion
of the cuttings in either NAA or IBA increased rooting percentage and root numbers
(Mackay and Kitto, 1998).
Liu et al., (2002) regenerated new shoots from hypocotyis in Artemisia
judaica L. via callus on medium supplemented with TDZ (thiadiazuron). Upto 16
shoots formed per seedling for an exposure of 20 days. Regenerated shoots formed
roots when subcultured onto a medium containing indole-3-butyric acid. The
regeneration protocol developed in this study provides a basic knowledge for
germplasm preservation and for further investigation of medicinally active
constituents of A. judaica.
Wang and Tan (2002) modified MS medium for enhancing Artemisinin
production in Artemisia annua hairy root cultures. They altered the ratio of NO3/NH4+ and the total amount of initial nitrogen. Increasing ammonium to 60mM
decreased both growth and Artemisinin accumulation. With NO3-/NH4+ at 5:1 (w/w),
the optimum concentration of total initial nitrogen for artemisinin production was
20mM. After 24 days of cultivation with 16.7mM nitrate and 3.3mM ammonium, the
maximum artemisinin production of hairy roots was about 14mg/L, a 57% increase
over that in the standard MS medium.
Nin et al (1996) initiated micropropagated A. annua L. plantlets on MS basal
medium supplemented with different concentrations of BA, Kin, NAA, IAA, and 2,4D alone or in combination. Supplementing the medium with low doses of both BAP
in combination with NAA, and Kinetin in combination with NAA enhanced the
growth rate of callus cultures. Initiation of root and shoot primordial directly from leaf
explants cultured on 1.81 M 2,4-D, while adventitious shoot formation from callus
was observed occasionally when BA was added to the medium in combination with
IAA. Furthermore, medium containing 2.22 M BA and 2.69 M NAA stimulated
both callus growth and organogenesis on some callus cultures derived from leaves and
stems of young stock material. The best results were obtained with leaf explants.
Cytological analysis of root meristems revealed that all regenerants were diploid
(2n=18), as expected.
1.7.4: Transformation
Transformation technology can be used to introduce genes to produce new
phenotypes (Hartwell et al., 2008) or to enhance the expression of existing genes
(Royal society, 2002). Effects have been made to enhance the production of
artemisinin in Artemisia dubia and Artemisia annua through transformation. The most
commonly used method of transformation in plants is Agrobacterium based.
1.7.4.1: Agrobacterium-mediated transformation
Agrobacterium tumefaciens has played a major role in the development of
plant genetic engineering and the basic research in molecular biology. It accounts for
about 80% transgenic plants produced so far. Initially, it was believed that only
dicots, gymnosperms and a few monocot species could be transformed by this
bacterium, but recent achievements totally changed this view by showing that many
―recalcitrant‖ species not included in its natural host-range such as monocots and
fungi can now be transformed (Chan et al., 2004; Bundock et al., 2005). In addition,
the transformed cells usually carry single or low copy number T-DNA integrated in
their genome with less rearrangement, and very large DNA segments can be
transformed into the plants (Liu et al., 1999).
1.7.4.1.1: Classification of Agrobacterium
The genus Agrobacterium has been divided into a number of species.
However, this division has reflected, for the most part, disease symptomology and
host range. The two most commonly used species for transformation purposes are
Agrobacterium tumefaciens and Agrobacterium rhizogenes (Stanton and Gelvin,
2003).
1.7.4.1.2: Agrobacterium tumefaciens
Agrobacterium tumefaciens is the soil born bacterium which, when containing
the Ti plasmid, is able to form crown galls on a number of dicotyledonous plant
species (Brown, 2001). Transformation is brought about by the transfer of a large
segment of the Ti plasmid called T-DNA to the nuclear genome of a susceptible plant
(Chilton et al., 2003). T-DNA contains genes for growth regulator autonomy
(Garfinkel et al., 2006 and Joss et al., 1989), and for the synthesis of a wide variety of
opines (Murai and Kemp, 1988) which are noble metabolites able to catabolishd by
the inciting Agrobacterium (Fig 1.9).
Figure 1.9: Structure of the Ti-plasmid of A.tumefacienes (taken from Hooykaas
and Schilperoort, 1992)
1.7.4.1.3: Agrobacterium rhizogenes
Agrobacterium rhizogenes is a natural plant pathogen responsible for
adventitious root formation at the site of infection (Hooykaas, 2004). It incites a
disease, hairy root disease, in dicotyledonous plants in a manner very similar to A.
tumefaciens (Old and Primrose, 1995). This morphogenic effect of A. rhizogenes is
due to the integration and expression of T-DNA of the Ri (root inducing) plasmid in
the plant cell genome (Willmitzer et al., 2006). It is also responsible for pathogenicity
and the induction of opine synthesis (White and Nester, 2003; Chilton et al., 1982).
The neoplastic roots are characterized by a high growth rate and are able to synthesize
some secondary metabolites (Flores and Filner, 2007).
The Ri plasmid share little homology with Ti-plasmids. The main difference
between Ri and Ti plasmid is that transfer of the T-DNA from a Ri plasmid to a plant
results not in a crown gall but in hairy root disease, typified by a massive proliferation
of a highly branched root system (Brown, 2001) (Fig 1.10). They are of interest
because tissue transformed by A. rhizogenes readily regenerates into plantlets, which
continue to synthesize opine. For these reasons transformed roots of many medicinal
and aromatic plants have been widely studied for the in vitro production of secondary
metabolites (Hamill et al., 1986; Benjamin et al., 1993; Jung and Tepfer, 1987; Mano
et al., 2005).
Figure 1.10: Structure of the Ri-plasmid of A.rhizogenes (taken from
Sheela, 2012)
Great progress has been made in recent years in studies on the mechanism of
Agrobacterium-mediated transformation and its application. Many details of the key
molecular events within the bacterial cells involved in T-DNA transfer have been
elucidated, and it is notable that some plant factors which were elusive before are
purified and characterized (Wei et al., 2000).
1.7.4.2: Molecular mechanism of Agrobacterium tumefacienes mediated
transformation
The mechanism of T-DNA processing and transfer during Agrobacteirum
infection has been subjected to a number of excellent reviews (Knight et al., 2010;
Kunitake et al., 2011). Principally, the bacterium can transfer a piece of its plasmid
DNA into the infected plant cells, where it integrates into the nuclear genome and
expresses its own genes, whose products disrupt the hormonal balance within the
plant cells and induce their proliferation to form tumors. In addition, it also produces
enzymes to synthesize opines, which the bacteria can use for their own nutrition. The
T-DNA is located on a large plasmid called Ti (tumor-inducing)-plasmid, which also
contains other functional parts for virulence (vir), conjugation (con) and the origin of
its own replication (ori). In the natural infection by wild type bacteria, the T-DNA and
the vir genes are essential for inducing plant tumors.
The vir region is about 30 kb and encodes at least 10 operons (virA-virJ)
whose products are vital to T-DNA processing and transfer. Any genes located in the
T-DNA region in principle can be transferred, but they themselves are dispensable for
this process. Only the 25-bp direct repeats at the right and the left borders are
necessary, of which 14 base pairs are completely conserved and cluster as two
separate groups. The infection begins at the wounded sites, and the injured plant cells
release some compounds such as the phenolic acetosyringone (AS) acting as specific
signals to bind and activate virA, a membrane protein, by autophosphorylation, which
subsequently activates VirG by phosphorylating one of its asparate residue. The active
form of virG binds specifically to the upstream of other vir genes called vir box,
inducing their expression. The virD1 and virD2 are responsible for the T-strand
generation, a single-stranded copy of the T-DNA, by specifically recognizing and
cutting the bottom strand at the two borders, of which the right one is the start site and
thus more important.
Figure 1.11: Transformation of plant cell by Agrobacterium tumefacienes
harbouring a wild-type Ti plasmid
After cutting, virD2 remains covalently attached to the 5' end of the T-strand,
forming a complex with, which is then targeted into the nucleus by the nuclear target
signals (NLSs) of its associated virD2 and virE2, where the T-DNA randomly
integrates into the plant genome as single or multiple copies. VirD2 has an active role
in the precise integration of T-strand the plant chromosome (Fig 1.11).
Novel strategies have to be developed for inserting new DNA into the plasmid.
Two are in general use:
1.7.4.2.1: The binary vector strategy is based on the observation that T-DNA does
not need to be physically attached to the rest of the Ti plasmid. A two-plasmid
system, with a T-DNA on a relatively small molecule, and the rest of the plasmid in
normal form, is just as effective at transforming plant cells. In fact some strains of A.
tumefaciens, and related Agrobacteria, have naturally binary plasmid systems. The TDNA plasmid is small enough to have a unique restriction site and to be manipulated
using standard techniques (White and Nester, 2003) (Fig 1.12).
Figure 1.12: The binary vector strategy. Plasmids A and B complement each
other when present together in the same A. tumefaciens cell. The T-DNA carried
by plasmid B is transferred to the plant chromosomal DNA by proteins coded by
genes carried by plasmid A.
1.7.4.2.2: The cointegration strategy uses as entirely new plasmid based on pBR322
or a similar E. coli vector, but carrying a small portion of the T-DNA. The homology
between the new molecule and the Ti plasmid means that if both are present in the
same A. tumefaciens cell, recombination can integrate the pBR plasmid into the TDNA region (Fig 1.13).
The gene to be cloned is therefore inserted into a unique restriction site on the
small pBR plasmid, introduced into A. tumefaciens cells carrying a Ti plasmid, and
the natural recombination process integrate the new gene into the T-DNA. Infection
of the plant leads to insertion of the new gene, along with the rest of the T-DNA, into
the plant chromosome (Brown, 2001).
Figure 1.13: The cointegration strategy
1.7.4.3: Production of transformed plants with Ti plasmid
As introduction of new genes into every cell in the plant is needed. For this
reason a culture of plant cells and protoplasts in liquid medium are infected. A mature
plant regenerated from transformed cells will contain the control gene in every cell
and will pass the control gene to its offspring.
However, regeneration of a transformed plant can occur only if the Ti vector
has been ―disarmed‖ so that the transformed cells do not display cancerous properties.
Infectivity is mainly controlled by the virulence region of the Ti plasmid i.e., two
25bp repeat sequences found at the left and right borders of the region integrated into
the plant DNA. Any DNA placed between these two repeat sequences will be treated
as T-DNA and transferred to the plant. A number of disarmed Ti cloning vectors are
now available; a typical example is the binary vector pBIN19. The left and right TDNA borders present in this vector flank a copy of the lac Z gene, containing a
number of cloning sites, and a kanamycin resistance gene that functions after
integration of the vector sequences into the plant chromosome. As with a yeast shuttle
vector, the initial manipulations that result in insertion of the gene to be cloned into
the pBIN19 are carried out in E. coli, the correct recombinant pBIN19 molecule then
being transferred to A. tumefaciens and then into the plant. Transformed plant cells
are selected by plating on agar medium containing kanamycin (Brown, 2001).
Several Agrobacterium strains and binary vectors have been used for the
production of transgenic Artemisia dubia and Artemisia annua (Vergauwe et al.,
1998; Nin et al., 2004).
1.7.4.4: Mechanism of A. rhizogenes infection
A.rhizogenes agropine type strains carry two T-DNAs, the TL-and the TRDNA (Jouanin, 1988). At least two pathogenic pathways, both leading to root
formation by the host, are realized by genes of the TL- and TR-DNA. The TR-DNA
harbors two auxin synthesizing genes, aux1 and aux2 (Camilleri and Jouanin, 1999).
The auxin biosynthetic pathway catalyzed by the aux1 and aux2 gene
products is similar to that in A. tumefaciens and Pseudomonas savastanoi and
comprises two steps. First, oxidative decarboxylation of tryptophan leading to indole3-acetamide (IAM) is catalyzed by tryptophan 2-monooxygenase (t2m), encoded by
the tms1, aux1, and iaaM genes of A. tumefaciens, A. rhizogenes, and P.savastanoi,
respectively (Camilleri and Jouanin 1999). The second step, the conversion of IAM to
indole-3-acetic acid (IAA), is catalyzed by IAM hydrolase, the product of the tms2,
aux2, and iaaH genes in the above bacteria (Jouanin, 1988).
In agropine type A. rhizogenes, the rol ABCD genes are primarily responsible
for root induction (White et al., 2008), possibly by enhancing the auxin sensitivity of
cells (Maurel et al., 2007; Spano et al., 2006). In some instances, rol B and additional
auxin act in concert to induce root formation; the factors alone are not effective
individually (Spena et al., 2002; White et al., 2008). TR-DNA genes could provide
this additional auxin if both T-DNAs are transferred. However, data indicate that TLDNA located genes in some hosts increase the auxin content, independent of the
presence of the TR-DNA. For example, it was shown that TL-DNA transformed pea
roots contain increased IAM and IAA concentrations (Prinsen et al., 1992).
Interestingly, iaam/tms1, gene expressing, transgenic asparagus and petunia
accumulate both IAM and IAA, while tms1-transformed tobacco accumulates only
IAM (Prinsen et al., 1998; Van Onkelen et al., 1985). This indicates that conversion
of IAM to IAA in cells that express only t2m depends on a host-specific factor such as
an endogenous hydrolase (Prinsen et al., 1992).
A candidate gene responsible for the increase IAM content in TL-DNA transgenic
tissue is the open reading frame 8 (ORF8). Transcripts possibly corresponding to
ORF8 have been reported for TL-DNA transformed tissues (Durand- Tardif et al.,
2006; Ooms et al., 2008), indicating that the gene is transcribed in infected cells (Fig
1.14).
Fig. 1.14: Process of hairy roots production through Agrobacterium rhizogenes
infection
1.8: Agrobacterium tumefacienes mediated transformation in Artemisia species
Agrobacterium tumefaciens mediated transformation has been used to
transform several crops as well as medicinal plants. Biswajit et al. (2000) established
transformed organ (petiole, lamina, node and internodes) cultures of the medicinally
important Artemisia annua following infection with two wild type nopaline strains of
Agrobacterium tumefaciens. Parameters such as explant type, strain type and age of
the explants source significantly affected tumorigenesis frequency. Crown galls were
formed both on in vivo and in vitro plants: 2-3% of the in vitro galls regenerated
spontaneously to produce shooty teratoma of altered phenotype. Artemisinin contents
were measured in all transformed as well as non-transformed clones. While shooty
teratomas synthesized 0.063 g/100 g DW artemisinin, non-transformed shoots
synthesized only 0.0179 g/100 g DW of the compound.
A transformation system was developed by Vergauwe et al. (2002a), for
Artemisia annua L. plants. Leaf explants from in vitro grown plants developed callus
and shoots on medium with 0.05 mg/L NAA and 0.5 mg/L BAP after transformation
with the C58C1 RifR (pGV2260) (pTJK136) Agrobacterium tumefaciens strain. A
concentration of 20mg/L kanamycin was added in order to select transformed tissue.
Kanamycin resistant shoots were rooted on NAA 1.0 mg/L. Polymerase chain
reactions and DNA sequencing of the amplification products revealed that 75% of the
regenerants contained the foreign genes. 94% of the transgenic plants showed a beta
glucuronidase-positive response.
In one study, Vergauwe et al. (2005), with a view to optimizing a previously
described Agrobacterium tumefaciens-mediated transformation procedure for
Artemisia annua, studied the importance of some parameters such as explants type,
age of explants source, A. tumefaciens strains and type of binary vector. Several
binary vectors were found useful for the production of transgenic callus on explants of
different ages. In transformed calli, a good correlation between integration and
expression of foreign DNA was observed: Different assays showed expression of
beta-glucuronidase, neomycin phosphotransferase II, superoxide dismutase and
bleomycin acetyltransferase. The regeneration of transgenic plants required more
restricted conditions. Only with the pTJK136 vector could transgenic plants be
obtained from leaf and stem explants of 12-18 weeks old plants. Cocultivation of 48 h
seemed favourable for the regeneration of transgenic plants. Stable integration and
expression of the transgenes were also shown in the progeny.
In another study, Mannan et al. (2009) established a transformation method for
Artemisia absinthium plants. Different factors such as age and type of explants,
presence of ampicillin and kanamycin in Agrobacterium inoculum, concentration of
Agrobacterium, infection and co-cultivation time period of Agrobacterium, effects of
2,4-D, pH of co-cultivation medium and effects of cefotaxime in regeneration
medium significantly affected transformation of Artemisia absinthium.
1.9: Agrobacterium rhizogenes mediated transformation in Artemisia species
Although artemisinin was thought to accumulate only in the aerial part of
Artemisia plants (Wallaart et al., 1999), several laboratories have shown that hairy
roots can produce artemisinin (Weathers et al., 2004; Jaziri et al., 1995; Liu et al.,
1999). Four Asteraceae species were tested: Artemisia dubia, Callendula officinalis,
Mikania glomerala and Helianthuus annuus. The explants of all species were
inoculated in Agrobacterium rhizogenes strains 8196 and 15834. Artemisia dubia and
Artemisia officinalis showed a positive hairy root response. These roots were excised
and cultured in vitro. In order to confirm the transgenic character of the hairy roots
Southern blot hybridization was carried out (Pellegrino et al., 2008).
Transformed cultures of Artemisia annua L. (Asteraceae) were established by
the co-culture method using leaf segments of A. annua and Agrobacterium rhizogenes
NCIB 8196 or MAFF 03-0172 by Jaziri et al., (1995), the hairy root clones grew
vigorously on hormone free medium. The genetic transformation of the root was
proved by the opine assay. A highly specific and sensitive enzyme-linked immunosorbent assay (ELISA) method was used for the detection and semi-quantitative
determination of artemisinin and structurally related compounds in these cultures.
Transformed root cultures of several strains of Artemisia annua were also obtained by
infection with Agrobacterium rhizogenes ATCC 15834 and found to be positive for
accumulation for artemisinin (Weathers et al., 2004).
Naturally, artemisinin accumulates in glandular trichomes, present in leaves,
small green stems, flowers buds and seeds of Artemisia (Martinez and Staba, 1988;
Ferreira et al., 1997). Although biosynthesis of artemisinin seems to be restricted to
the green parts of the plants, and is not synthesized in roots of field-grown plants
(Ferreira et al., 1995) or pollens, yet it can be produced by transformed roots of
Artemisia dubia and Artemisia indica as described by Mannan et al., (2008).
According to Nin et al., (1997), hairy roots were obtained after infection of
Artemisia annua shoots with Agrobacterium rhizogenes strains 1855 and LBA 9462.
The susceptibility to hairy root transformation varied between plant genotypes and
bacterial strains. Hairy roots showed macroscopic differences from control root
cultures. Southern blot hybridization confirmed the integration of T-DNA from both
p1855 and pBin19, while polymerase chain reaction analysis indicated the presence of
the NptII gene in the hairy root genome. Sub-cultured transformed root lines grew
well in selective B5 agar-solidified medium containing kanamycin or rifampicin and
without hormones. Great differences were found in the profiles of the essential oils
isolated from normal and hairy roots.
1.10: Role of rol genes in plants
Agrobacterium rhizogenes and Agrobacterium tumefaciens infect wounds of
several dicotyledonous and some monocotyledonous species and thereby generate
tumorous outgrowths at the site of infection (Nilsson and Olsson, 1997). The genes
required for tumorigenesis are found on extra-chromosomal elements, Ti (tumor
inducing) plasmids for A. tumefaciens and Ri (root inducing) plasmids for A.
rhizogenes, of which a T-DNA (transferred DNA) portion is integrated into the plant
genome (Zupan et al., 1995; Zhu et al., 2000). Both pathogens are able to re-specify
differentiated cells to gain meristematic functions and, in case of A. rhizogenes,
infections are characterized by a massive production of adventitious roots (Meyer et
al., 2002).
Rol genes have tremendous effect on production of secondary metabolites.
Transformation with the rol A gene results in plants with a highly aberrant phenotype,
characterized by wrinkled, intensely green leaves, long internodes, dwarfism or semi
dwarfism and retarded senescence (Altvorst et al., 1992; Schmulling et al., 1993). The
pleiotropic alterations observed in tobacco and potato has led to the hypothesis of a
functional imbalance in the auxin/cytokinin ratio in favour of cytokinins (Schmulling
et al., 1993). In transgenic tobacco plants a reduction of gibberellic acid content has
also been reported, suggesting the involvement of rol A in gibberelin metabolism
(Dehio et al., 1998).
The rol B protein on the other hand, has been shown to have a tyrosine
phosphatase activity and therefore a possible role in the auxin signal transduction
pathway (Filippini et al., 1994). Artemisia plants transgenic for rol B have wider
leaves and a reduced apical dominance than the wild type (Altvorst et al., 1992). The
rol B gene introduced in Nicotiana tabacum was responsible for an auxin like activity,
such as enhanced adventitious root formation, which is correlated with increased
auxin sensitivity (Maurel et al., 1991; Filippini et al., 1994; Maurel et al., 1994).
Similar effect of rol B was also observed in tobacco leaf explants and thin cell layers
by Altamura et al. (1998). Hairy roots from different species (Shen et al., 2001; Shen
et al., 2003) as well as leaf fragments from hairy root tobacco regenerants (Spano et
al., 1988) were shown to be more sensitive to auxin than their normal counterparts.
This feature suggests that Ri T-DNA genes induce the proliferation of transformed
cells by a unique mechanism, as compared to A. tumefaciens oncogenes, which cause
disease by encoding enzymes for hormone biosynthesis (Zambryski et al., 1989).
The rol C involved in the released of active cytokinins from their inactive
glucosides due to its cytokinin glucosidase activity Estruch et al. (1999). This role is
consistent with the observed phenotype of rol C transgenic plants, characterized
mainly by a reduction of apical dominance and plant height (Estruck et al., 1999;
Nilsson and Olsson, 1997, Schmulling et al., 1993). The integration and expression of
the T-DNA of Ti plasmids causes metabolic changes mainly determined by the iaa M
and iaa H genes which code for enzymes involved in indoleacetic acid synthesis and
the ipt gene whose product catalyses the first step in cytokinin biosynthesis (Yamada
et al., 1985). In ornamental plants such as carnation and Petunia the insertion of rol C
leads to the expression of advantageous traits, i.e., increased axillary bud break and
development, better rooting ability of stem cuttings (Zuker et al., 2001), increased
branching and reduction in time to flowering (Winefield et al.,1999). Some data
reveals that the rol C gene could affect activity of defence proteins, such as 1, 3-Dglucanases. Expression of the rol C gene in ginseng cells caused significant increase
of 1, 3-D-glucanase activity by production of a new enzyme isoform. Activities of
other glucanases tested such as 1, 4-D-glucanases and 1, 6-D-glucanase, which are
known to be unrelated to defence proteins, were unchanged (Bulgakov et al., 2002).
Concerning the rol D gene, its function as an ornithine cyclodeaminase
enzyme, catalyzing, catalyzing the conversion of ornithine to proline, has recently
been elucidated (Trovato et al., 2001). In transgenic tobacco plants, rol D induces a
striking earliness in the induction of the flowering process and an increase in the
number of flowers (Mauro et al., 1994) that have been related to an accumulation of
proline or to a depletion of ornithine. In Artemisia plants rol D has a pleiotropic
effect, affecting traits of economic interest such as plant productivity, as well as
characters generally correlated with the defence response to pathogens (Bettini et al.,
2003). In this study, the analysis of transgenic plants did not show any morphological
modifications. First generation of transgenic plants was found to flower earlier, and
showed an increased number of inflorescence and higher fruit yield. They have also
shown that the plants harbouring rol D gene were more tolerant to the toxin produced
by the fungus fusarium oxysporum in ion leakage experiments, with respect to the
untransformed regenerated controls.
The organogenetic competence of roots and Agrobacterium rhizogenes
induced hairy roots of twelve Artemisia genotypes has been investigated (Peres et al.,
2001). Both roots and hairy roots of Artemisia dubia L. derived genotypes regenerated
shoots after 24 weeks of incubation on zeatin contained medium. Anatomical analysis
showed that shoot regeneration in roots could be direct or indirect, depending on the
genotype considered. Hairy roots showed considerable differences in their
morphogenetic responses, when compared to the corresponding non-transgenic roots.
The differences observed might reflect the influence of the introduced rol genes on
hormonal metabolism/sensitivity. Data pointed to the possibility of the use of A.
rhizogenes, combined with regenerating Artemisia genotypes, in a very simple
protocol, based on genetic capacity instead of special procedures for regeneration, to
produce transgenic Artemisia plants expressing rol genes, as well as, genes present in
binary vectors.
Root proliferation is not due to diffusible cell division factors and a direct
interaction of the proteins encoded on the T-DNA with plant hormonal metabolism
could not be shown. The T-DNA encodes up to 18 open reading frames, depending on
the bacterial strain (Slightom et al., 1986). Insertional mutagenesis showed that
insertions in only 4 of the potential 18 loci noticeably affected the morphology of the
hairy roots that were produced (White et al., 1985). These loci were named root locus
A-D (rol A-d). The rol A, B, C and D genes have been identified as the main
determinants of the hairy root disease caused on dicotyledonous plants by the soil
bacterium Agrobacterium rhizogenes. The rol A, B, C and D loci correspond, most
likely, to open reading frames (ORFs) 10, 11, 12 and 15 of the TL-DNA (Slightom et
al., 1986). When individual rol genes are inserted in plants, they have different
phenotypic effects that can be at least in part ascribed to modifications in the
endogenous hormone equilibrium and in some cases have shown to be potentially
interesting also for economic purposes.
The roots induced by the integration of T-DNA of various Agrobacterium
rhizogenes wild type strains are characterized by an extensive growth, associated with
lateral branching, leading to an important mass of adventitious roots exhibiting a
typical phenotype. Furthermore, these roots has been found to produce a high yield of
secondary metabolites as compared to that of undifferentiated plant cell suspensions,
such as indole alkaloid production in Catharantus roseus cultures (Palazon et al.,
2003), ginsenoside production in cultures of Catharatus roseus cultures (Palazon et
al., 2003), ginsenoside production in cultures of Panax ginseng (Bulgakov et al.,
2002), and anthraquinone production in callus cultures of Rubia cordifolia (Bulgakov
et al., 2002). The hairy roots obtained from the transformation of Atropa belladonna
with A. rhizogenes 15834 produced various amounts of tropane alkaloids, and in most
cases higher than in normal field grown plants (Kamada et al., 1986; Saito et al.,
2001). The rol A, rol B, and rol C genes together induced root formation and tropane
alkaloid biosynthesis in tobacco and in Datura stramonium (Spena et al., 2002;
Palazon et al., 1997). The rol A, B and C genes could play a major role in hairy root
induction and metabolite production, also leading to an increase in the production of
secondary products in A. belladonna hairy roots as is the case with tobacco root
cultures (Palazon et al., 1997). The capacity to induce root formation in the host is
greatly increased when the rol A and/or C loci are combined with the rol B locus.
Root induction is shown to be correlated with the expression of the rol loci.
Transgenic plants exhibit all the characteristics of the hairy root syndrome only when
all three loci are present and expressed. Although the activity of the rol genes encoded
functions is synergistic, each of them appears to independently influence 3 host
functions involved in the determination of root differentiation (Spena et al., 2002).
1.10.1: Synergistic effects of rol genes
A number of synergistic effects of rol genes have been reported. The presence
of rol A, rol B and rol C together dramatically enhances the growth rate and nicotine
production, which shows that the effect of these three rol genes was synergistic
(Plazon et al., 1997). Several reports suggest that rol A and rol C alter Polyamine
(PA) metabolism and plant phenotype (Michael et al., 1998; Sun et al., 1999; MartinTanguy et al., 2006). In another study, transgenic plants containing the rol A
constructs showed an increase in the level of PA biosynthetic enzymes and PA titres
(Altabella et al., 1995). It appears that the degree of expression of rol A and/or rol C
phenotype depends on the level of their penetration, type of tissue used, promoter
strength and site of insertion of the foreign gene into the plant genome. Transgenic
roots, especially those transformed either by a combination of the three rol genes
(A+B+C) or the rol C alone, grew faster than the untransformed roots (Altabella et
al., 1995). Putrescine, spermidine and traces of spermine were present in all samples,
both in free and bound forms, while rol A roots showed increased levels of free and
bound polyamines (Altabella et al., 1995). The higher polyamine contents found in
roots transformed by rol A paralleled with higher ornithine and arginine
decarboxylase activities as well as higher nicotine production (Altabella et al., 1995).
Little is known about mechanisms by which the rol genes interact secondary
metabolism. Studying the transgenic for the rol B and rol C genes callus cultures of R.
cordifolia, it has been found that anthraquinone (AQ) production was greatly
increased in both transformed cultures compared with the non-transformed cultures
(Bulgakov et al., 2002). The induction of AQ biosynthesis by the rol genes did not
proceed through the activation of the common Ca+2 –dependent NADPH oxidase
pathway that mediates signal transduction between an elicitor receptor complex via
transcriptional activation of defence genes (Bulgakov et al., 2003). Okadaic acid and
cantharidin, inhibitors of protein phosphatases 1 and 2A, caused an increase of AQ
production in transgenic cultures. Okadaic acid stimulated AQ accumulation in the
non transformed cultures, whereas cantharidin had no effect. These results have
shown that different phsophatases are involved in AQ synthesis in normal and
transgenic cultures of R. cordifolia (Bulgakov et al., 2003).
1.11: Extraction of artemisinin
Conventionally, Chinese extracted the artemisinin from different parts of
Artemisia plants with hot water by using common rules of extraction of Chinese
medicine (Stoger, 1991). Researchers have developed many methods to extract
artemisinin from plant material, on small (Kim et al., 2002; Liu et al., 2007) as well
large scale (ElSohly et al., 1990). Extraction can be done from fresh (Filip et al.,
2006) as well as dried plant parts (Widmer et al., 2007) with equal efficiency. Ferreira
and Janick, (2002) described that artemisinin can be easily extracted with petroleum
ether, which boils from 30 to 60°C or other solvent such as chloroform, acetonitrile
and ether, which have a boiling point lower than critical temperature for artemisinin
stability. Artemisinin can remain stable up to 150°C in neutral solvent as explained by
Lin et al. (1985). At the moment, extraction of artemisinin on large scale is carried out
by using the methods described by Klayman et al. (1984). This method includes the
extraction of artemisinin from dried leaves of Artemisia annua by using petroleum
ether and fractionated on a silica column. Another method which is very economical
depends upon the use of multiplayer separator and extractor (Acton et al., 1986) but it
is only suitable for small-scale extraction.
Large amount of artemisinin was extracted by Elsohly et al., (1987), but
artemisinic acid tends to elute with artemisinin and the fraction containing artemisinin
may require rechromatography to achieve the necessary purity. To resolve this issue,
Elsohly et al. (1990) described a practical and economical procedure for the isolation
of artemisinin in kilogram quantities by extracting leaves with hexane followed by
partitioning with a methyl cyanide phase. This method produced 99% pure
artemisinin without any contamination of artemisitene. The common extraction
methods for artemisinin neglects artemisinic acid, but an efficient method for
extraction of both artemisinic acid and artemisinin from the same material was
proposed by Vonwiller et al. (1993) which can then be converted to artemisinin (Xu
et al., 1986) that significantly enhanced final yield of artemisinin. However, a ton of
dried leaves are needed to produce about 6kg of artemisinin.
1.12: Analysis of artemisinin
Artemisinin is an unstable compound and found in very low concentration in
plants, therefore its analysis is very complicated because the intact molecule stains
poorly, and other compounds interfere in its detection in crude plant extracts.
Although artemisinin is very stable even at 150°C but when heated at 180-200°C,
degrades into several products (Lin et al., 1985; Luo and Shen, 1987) and it is also
sensitive to acid and base treatment (Zheng et al., 1983).
Many scientists used thin later chromatography for rapid estimation of
artemisinin content in A. annua (Tu et al., 1982; Klayman et al., 1984; Luo and Shen,
1987; Roth and Acton, 1985; Tawfiq et al., 1989; Pras et al., 1991) but this method is
not very reliable due to poor staining characteristics of intact molecule and
contamination with other constituents of the plant.
Many laboratories used HPLC with UV monitoring at 210 nm but presence of
constituents that absorb UV at this wavelength completely obliterate the peak of
artemisinin (Acton et al., 1985; Zhao and Zeng, 1985; Liersh et al., 1986; Pras et al.,
1991), due to this reason some researchers used absorbance range of 220 nm for
artemisinin detection (Dhingra et al., 2000). At the moment, many researchers used
absorbance UV range of 260 nm in HPLC for artemisinin detection and this is most
authentic, economical, practical and extensively used method for the quantification of
artemisinin (Smith et al., 1997; Kim et al., 2001; Towler and Weathers, 2007).
Reversed phase HPLC is also very useful for the determination of artemisitene
in artemisinin, this method can also be used to check the purity level of artemisinin,
and for preparative scale purification of these compounds as reported by Eldomiaty et
al. (1991). Detection of artemisinin and its derivatives by using HPLC method with
polarographic detection has also been used by Zhou and Xu (1988).
Variety of methods have been suggested for the analysis of artemisinin; for
example Fulzele et al., (1991) and Sipahimalani et al., (1991) used gas
chromatographic (GC) method for the analysis of artemisinin at nanograms levels
(detection limit 100 ng). Other methods include rapid screening based on mass
spectrometry (MS) used for artemisinin related compounds present in crude hexane
extract of A. annua (Ranashige et al., 1993). In addition, there are some reports
proving the detection of artemisinin in which GC combined with MS (Banthorpe and
Brown, 1989; Woerdenbag et al., 1991; 1993) as well as detection by
radioimmunoassay or RIA (Zhao et al., 1986) is used. Jaziri et al. (1993) reported the
use of enzyme-linked immunosorbent assay (ELISA) method to detect the artemisinin
and closely related compounds in crude extract of A. annua.
1.13: Biosynthetic pathway of artemisinin
A lot of work has been done to understand biosynthetic pathway of artemisinin.
Terpene biosynthesis involves two independent and differently localized mechanisms.
Both pathways converge to produce a terpenoid precursor isopentenyl diphosphate
(IPP) (Croteau et al., 2000; Lange et al., 2000) (Figure 1.15).
The mevalonic acid pathway (MVA) which initiates from acetyl-CoA is located
in the cytosol. The key, regulatory, step in this pathway is the conversion of
hydroxymethylglutaryl-CoA (HMG) to mevalonate via, a regulatory enzyme,
hydroxymethylglutaryl-CoA reductase (HMGR). Several subsequent steps lead to
formation of the cytosolic localized pool of IPP (Towler and Weathers, 2007).
The other pathway, non-mevalonate pathway (MEP), to IPP begins with pyruvate
and occurs in the plastid with no mevalonate intermediate. The first key, regulatory,
step is the synthesis of 1-deoxy-D-xylulose-5-phosphate (DXP) via 1-deoxy-Dxylulose-5-phosphate synthase (DXS). DXP is then converted to 2-C-methyl-Derythritol-4-phosphate
via
1-deoxy-D-xylulose-5-phosphate
reducto-isomerase
(DXR). This is the first committed MEP step towards synthesis of terpenes. Several
subsequent steps synthesize the plastid pool of IPP (Rohmer et al., 1996).
Although IPP present in the cytosol is generally used for the biosynthesis of
sterols, sesquiterpenes, triterpenes, and polyterpenes, and the IPP present in the
plastid is used for the biosynthesis of monoterpenes, diterpenes and carotenoids
(Towler and Weathers, 2007). Translocation of IPP from the cytosol to the plastid or
vice versa may occur depending on the needs of the plant (Adam et al., 1998; Lange
et al., 2000).
Evidence exists suggesting that the two compartmentalized pathways of terpene
biosynthesis may communicate with each other to regulate metabolic intermediate
availability. There are no absolute restrictions on the compartmentalization of
intermediates in the pathway and the degree of separation probably depends on the
species and physiological conditions (Hampel et al., 2005). In A. annua, evidence
suggests that both the MVA and MEP pathways seem to play a role in artemisinin
production. For example, when the cytosolic MVA pathway was disrupted by
inhibiting HMGR with mevinolin, artemisinin levels dropped by about 80 %. When
DXR in the MEP pathway was inhibited by fosmidomycin, artemisinin levels dropped
about 70 %. Use of both inhibitors resulted in no detectable artemisinin production
(Towler and Weathers, 2007).
1.13.1: Post-IPP terpene biosynthesis
Once IPP is formed and available in the cytosol, the next step towards
artemisinin biosynthesis is the production of farnesyl diphosphate (FPP) via farnesyl
diphosphate synthase (FPS). Sequence analysis of FPS from A. annua has shown a
very close similarity to FPS from other plants (Matsushita et al., 1996). Indeed
transgenic A. annua plants expressing FPS under a constitutive promoter produced 34 times more artemisinin than control lines, suggesting that FPS is one of the
regulatory points in artemisinin biosynthesis (Chen et al., 2000a). FPP is a branch
point, leading to the biosynthesis of triterpenes, polyterpenes, sterols, and
sesquiterpenes (Fig 1.15).
Figure 1.15: Early biosynthetic pathway for artemisinin: steps in the MVA and
MEP pathways and post-IPP steps (taken from Towler and Weathers, 2007)
There is evidence suggesting that in plants the option to branch towards either
sterols or sesquiterpenes is under coordinate control. To produce sesquiterpenes, a
sesquiterpene cyclase (SQC) is the required first catalyst; to produce sterols, the
required first catalyst is a specific squalene synthase (SQS). In plants, when sterol
production is upregulated, sesquiterpene production is often down regulated, and vice
versa. Vogeli and Chappell, (1988) demonstrated that introducing fungal elicitors to
tobacco cell culture suspensions caused a rapid increase in sesquiterpenoid production
paralleled by a rapid decrease in sterol production. This coordinate control of SQS
and SQC genes has also been demonstrated in potato tubers (Yoshioka et al., 1999;
Krits et al., 2007). Upon wounding, metabolic flow is directed towards sterols by the
up regulation of SQS and downregulation of SQC.
On the other hand, exposure of the wound to fungal pathogens or elicitors
causes redirection of metabolic flow towards sesquiterpenoid phytoalexins and SQC
is upregulated while SQS is down regulated. In A. annua, Towler and Weathers,
(2007) showed that coordinate control of these two pathways may be in play.
Inhibition of SQS with miconazole caused a significant increase in artemisinin,
suggesting that carbon was channelled towards sesquiterpene synthesis once sterol
biosynthesis was inhibited.
1.13.2: Committed steps in artemisinin biosynthesis
The first committed step in artemisinin biosynthesis (Fig. 1.16) is the cyclization
of farnesyl diphosphate (FPP) to generate amorpha-4, 11-diene, catalysed by
amorpha-4, 11- diene synthase (ADS) (Mercke et al., 2000; Wallaart et al., 2001).
Subsequent oxidation at the C12 position, mediated by the cytochrome P450 enzyme
CYP71AV1, leads to artemisinic alcohol (Ro et al., 2006; Teoh et al., 2006). While
arteannuin B has been suggested as a late precursor in artemisinin biosynthesis
(Sangwan et al., 1993; Zeng et al., 2008), evidence now favours a route from
artemisinic alcohol via dihydroartemisinic acid (Bertea et al., 2005; Covello et al.,
2007; Covello, 2008).
This route is supported by the cloning and characterization of double bond
reductase 2 (DBR2), which reduces the D11 (13) double bond of artemisinic
aldehyde, but not of arteannuin B (Zhang et al., 2008), and the cloning of aldehyde
dehydrogenase 1 (ALDH1), which catalyses the oxidation of artemisinic and
dihydroartemisinic
aldehyde
(Teoh
et
al.,
2009).
The
conversion
of
dihydroartemisinic acid to artemisinin, and of artemisinic acid to arteannuin B, has
been suggested to occur via enzyme-independent reactions (Sy & Brown, 2002;
Brown & Sy, 2004, 2007). Recently, a broad substrate oxidoreductase (RED1) with
high affinity for dihydroartemisinic aldehyde and monoterpenes was identified that
may have a negative impact on the flux to artemisinin biosynthesis (Ryde´n et al.,
2010).
IPPI
DMAPP
FDS (2X IPP+ DMAPP)
IPP
Farnesyl Diphosphate (FPP)
Amorpha-4, 11-dien3
CYP71AV1
Artemisinic Alcohol
CYP71AV1
Dihydroartemisinic Alcohol
CYP71AV1/ALDHI
RED1
DBR2
Artemisinic Aldehyde
Dihydroartemisinic Aldehyde
CYP71AV1/ ALDHI
Artemisinic Acid
ALDH1
Dihydroartemisinic Acid
Arteannuin B
Artemisinin
Fig. 1.16: Biosynthetic pathway of artemisinin (taken from Lies et al., 2011)
1.13.3: Regulation of the artemisinin biosynthetic pathway
Although many of the genes involved in artemisinin biosynthesis have been
isolated and cloned from A. annua, little is known about their regulation. Most of the
data relates to the effects of light, culture age, and tissue location on the expression of
these genes with most results to date measured in hairy root cultures (Souret et al.,
2003; Teoh et al., 2006).
The shift from vegetative growth into reproductive growth of A. annua is another
factor that has been shown to influence artemisinin production (Ferreira et al., 1995b).
Since many terpenoids are floral fragrances, up-regulation of terpene biosynthesis
during the shift from vegetative to flowering is not surprising. Previous studies had
suggested that a link between flowering and artemisinin biosynthesis exists, although
these studies differed in relation to the stage during which peak artemisinin
production was reached. One study, for example, showed that peak artemisinin
production occurs in the budding stage just before flowering (Liersch et al., 1986;
Woerdenbag et al., 1994; Chan et al., 1995), while others reported that peak
production was only reached when the flowers were in full bloom (Ferreira et al.,
1995b). Some genes related to terpene synthesis in other plants have also been shown
to be transcriptionally activated as the shift to flowering occurs (Dudareva et al.,
2003).
More recently, two separate studies investigated the link between flowering
and artemisinin. The flowering promoter factor genes from Arabidopsis, fpf1, and the
early flowering gene from Arabidopsis, CONSTANS, were constitutively expressed
in A. annua, and although flowering was induced approximately two to three weeks
earlier in transgenic lines, there was no corresponding increase in artemisinin
biosynthesis. These data suggested that there was no direct regulatory link between
flowering and artemisinin synthesis, and that some other factor is likely contributing
to the observed increase in artemisinin content as the shift to the reproductive stage
progresses (Wang et al., 2004; Wang et al., 2007).
Numerous investigations have shown that the Agrobacterium rol genes can
induce high levels of secondary metabolites in hairy root cultures of most transformed
plant species (Giri and Narasu, 2000; Sevon and Oksman-Caldentey, 2002). We
hypothesized that transformation with the rol genes could induce an increase in
artemisinin through stimulation of the synthesis pathways. Hence, the aim of the
investigation reported here was to compare artemisinin production in transformed
plants and hairy roots of Artemisia dubia and Artemisia annua transformed with rol
genes by using Agrobacterium tumefaciens and Agrobacterium rhizogenes with non
transformed plants to determine their effect. Trichome densities and the genes
involved in the synthesis pathway were monitored to ascertain how the rol genes act
to influence the production of artemisinin.
1.14: Aims and objectives
The following were the main objectives of this study:
1: To optimize and develop the standard transformation procedures by Agrobacterium
tumefacienes and Agrobacterium rhizogenes in Artemisia annua and Artemisia dubia.
2: To compare and analyse artemisinin and artemisinin derivatives contents of
transformed and non-transformed plants of A. annua and Artemisia dubia.
3: To monitor the metabolic pathway of artemisinin and the genes involved in the
synthesis pathway to ascertain how the rol genes act to influence the production of
artemisinin in shoots, roots and hairy roots of Artemisia annua and Artemisia dubia.
4: To compare the trichome densities in shoots, roots and hairy roots of transformed
and untransformed plants of Artemisia annua and Artemisia dubia and to monitor
their effect on artemisinin production.
5: To analyse the anticancerous properties of transformed and non-transformed plants
of Artemisia annua and Artemisia dubia.
6: To study the transcriptome of Artemisia annua and Artemisia dubia through
sequencing, their comparison in transformed and control plants and to study the
homology of their genome sequence.
Chapter No. 2
Genetic Transformation of Artemisia annua and Artemisia dubia with rol genes through
Agrobacterium tumefacienes and Agrobacterium rhizogenes
2.1: Introduction
Artemisinin and its derivatives are used in combination therapies for the
treatment of malaria (Haynes, 2006), and for treatment of numerous cancers and viral
diseases (Efferth, 2007; Efferth et al., 2008). However, artemisinin and its derivatives
are not available to the millions of the world‘s poorest people because of the low yield
of artemisinin in naturally growing Artemisia plants (0.1% to 0.5%) of dry weight
(Wallaart et al., 1999). The enhanced production of artemisinin, therefore, is highly
desirable. Genetic improvement of natural varieties has been attempted but the
maximum yield of artemisinin from this route reached so far is 2% (Graham et al.,
2010). Through transformation we can enhance the production of artemisinin in
Artemisia dubia and Artemisia annua by inserting our desired genes.
Plant cells transformed with the rol genes have been observed to show
increased production of secondary metabolites. Production of transgenic plants seems
to be the most appropriate choice to improve production of secondary metabolites of
any plant. For this purpose Agrobacterium mediated transformation method is being
used for several medicinal plants including Artemisia species (Nin et al., 1996; Nin et
al., 1997; Vergauwe et al., 1998). Optimized conditions may provide a mean to
improve the introduction of foreign genes into Artemisia species. Several
Agrobacterium strains have been used for the production of transgenic Artemisia
annua (Vergauwe et al., 1996; 1998) and Artemisia absinthium (Nin et al., 1997).
Vergauwe et al., (1996) developed a transformation system for Artemisia annua by
using Agrobacterium tumefaciens strain C58C1 RifR (pGV2260, pTJK136).
The aim of present study was to optimize the transformation conditions for
A.annua and A.dubia, establishment of hairy root cultures and production of different
transgenic lines of both species. Molecular analysis was performed for confirmation
of transformation through PCR and Southern blot analysis. A number of factors have
been studied like type of explants, different Agrobacterium strains, sterilization
conditions and co-cultivation period affecting transformation conditions of Artemisia
annua and Artemisia dubia by using Agrobacterium tumefaciens and Agrobacterium
rhizogenes. The conditions optimized in this study can be used later to transform other
Artemisia species in order to enhance the artemisinin content in plant.
2.2: Materials and Methods
The present research work was carried out in Plant Molecular Biology
laboratory, Department of Biochemistry, Quaid-i-Azam University, Islamabad and
Plant molecular biology lab, department of life sciences, Warwick University, United
Kingdom. A brief account of the materials and methods used and the procedures
adopted is given below.
2.2.1: Glassware and chemicals
Glassware used in all the experiments was made up of borosilicate (Pyrex).
All the glassware was cleaned by boiling in a saturated solution of sodium
bicarbonate for 1 hour followed by repeated washing in tap water. Thereafter, these
were immersed for 30 minutes in 30% HNO3 solution followed by repeated wash in
tap water. Washed glassware was further rinsed with distilled water and then dried at
200°C in an oven. Test tubes and flasks were plugged with absorbent cotton.
Autoclaving of the glassware was carried out at 121°C, 15 lbs psi for 30 minutes.
Chemicals used in all the experiments of tissue culture study were of
analytical and molecular biology grade procured from Sigma Chemical Co., USA.
Growth regulators and antibiotics (except cefotaxime) were also obtained from Sigma
Chemical Co., USA and Melford Laboratories Ltd, UK. Molecular biology products
were purchased from Sigma, Invitrogen and Fermentas while kits used were obtained
from Qiagen, Promega and MultiTarget Pharmaceuticals. Cefotaxime, sucrose,
glucose, gelling agent (gelrite), agar-agar and Hi-Media Bacto-Agar for microbial
work were procured from ―DIFCO‖ laboratories, USA.
2.2.1.2: Medium
Murashige and Skoog (MS) medium (Murashige and Skoog, 1962) (AppendixIII), (MS; Sigma cat# M5519) was used for germination of seeds in the present
investigation. MYA (Appendix-II) and YMB (Appendix-I) media were used for the
growth of bacteria.
2.2.3: Inoculation area and manipulation tools
Transfer room was cleaned on monthly basis and sprayed with 95% ethyl
alcohol. Surgical instruments, Petri dishes, flasks containing distilled water were
sterilized in an autoclave at 121 °C and 15psi for 20 minutes. Surgical instruments
and Petri dishes were autoclaved in aluminium foil or newspaper.
2.2.4: Culturing of tissues
Aseptic transfer of tissues was carried out in a Laminar Flow Cabinet fitted with a
HEPA filter. Before using the Laminar Flow Cabinet, working surfaces were swabbed
down with 95 % ethyl alcohol.
Surgical instruments, Petri dishes, distilled water and culture vessels were
brought into the cabinet. Then the working area and instruments were exposed to UV
light for 20 minutes to ensure sterility. Tissue culture work was started about half an
hour after the UV light was switched off. Surgical instruments were dipped in 95 %
ethyl alcohol. After each manipulation, the instruments were again dipped in ethyl
alcohol, reflamed and reused.
2.2.5: Collection of plant material
The plants and seeds of A.dubia Wall. and A.annua L. were collected from
Donga gali Ayubia pipeline (NWFP) and Astour (Northern areas) Pakistan, which
were identified by Taxonomy Lab, Department of Plant Sciences, Quaid-i-Azam
University, Islamabad, and were stored in refrigerator at 4 ◦C. These seeds keep their
vigor for at least 3 years if stored under dry and cool conditions.
2.2.6: Seed germination
Stored seeds of Artemisia dubia and Artemisia annua were surface sterilized
with 0.1 % (w/v) Mercuric chloride (HgCl2) solution for varying durations of 2, 3, 4
and 6 minutes. Then seeds were rinsed several times with sterile distilled water under
laminar flow hood. Seeds were sown under sterile conditions in Petri dishes
containing half strength MS salts, supplemented with 3% (w/v) sucrose and solidified
with 0.8% (w/v) agar. The pH was adjusted to 5.8 with 1 N NaOH / HCl before the
addition of agar. The medium was autoclaved at 15 psi, 121ºC for 20 minutes. This
medium is referred to as the Germination Medium (GM). Then these plates were
incubated in growth chamber at 25ºC 16 h of photoperiod, illumination of 45 µE m-2 s1
and 60 % relative humidity under aseptic conditions.
2.2.7: Culture environment
Tissue cultures were incubated under conditions of well-controlled
temperature, light intensity and photoperiod. Temperature of culture room for the
present study was maintained at 25 2 ºC with 16 / 8 hours light dark cycle and the
light intensity was maintained at 1000 lux throughout the experiments.
2.2.8: TRANSFORMATION OF ARTEMISIA SPECIES WITH ROL ABC
GENES
2.2.8.1: Agrobacterium tumefaciens mediated transformation of Artemisia dubia
and Artemisia annua with rol ABC Genes
The transformation procedure involving tissue culturing was carried out with
two different types of explants i.e. Stem and leaf with Agrobacterium tumefacienes
strain LBA4404 containing pRT99 harbouring rol ABC genes.
2.2.8.2: Plant material
Stem and leaves (0.5-1 cm in length) were cut transversely from 20 days –old in
vitro grown seedlings of both Artemisia species and used for genetic transformation.
In transformation experiment, 20-40 control explants were used without cocultivation
with Agrobacterium.
2.2.8.3: Bacterial strain and plasmid construction
Agrobacterium tumefacienes strain LBA4404 harbouring rol ABC genes was
kindly provided by Dr. David Tepfer, institute National de la Recherche (INRA),
Vesailles, France. T-DNA region of pRT99 carries rol A, rol B and rol C coding
sequences, each of which is under the control of CaMV 70S promoter and CaMV 35S
terminator. T-DNA of pRT99 also contains NPTII gene with NOS promoter and NOS
terminator (Fig 2.1).
Fig 2.1: Schematic diagram of the T-DNA region of the plasmid pRT99
RB: Right border; LB: Left border; NOS PRO: Nopaline synthase promoter; NOS
TER: Nopaline synthase terminator; 35SPRO: Cauliflower mosaic virus 35S
promoter; 35S TER: Cauliflower mosaic virus 35S terminator; 70S PRO:
Cauliflower mosaic virus 70S promoter; NPTII: Neomycin phosphortransferase
coding region.
2.2.9: TRANSFORMATION PROCEDURE
2.2.9.1: Preparation of explants
Leaves and stem segments, were excised from 20 days old seedlings of both
Artemisia species and cut into 0.5-1 cm pieces under laminar flow hood and
transferred to MS medium containing 200µM acetosyringone in petriplates. These
pieces were used as explants for further manipulation.
2.2.10: Co cultivation
2.2.10.1: Co cultivation medium
Co-cultivation medium was prepared by adding 200µM acetosyringone to MS,
pH was adjusted at 5.8. The medium was prepared in 500ml flasks which were
plugged with cotton, covered with aluminum foil and autoclaved at 15psi, 121ºC for
20 minutes. The autoclaved medium was poured under laminar flow hood, into Petri
plates (75mm) containing 25ml of medium in each. The media in the Petri plates was
allowed to solidify under the sterilized conditions of laminar flow hood.
2.2.10.2: Co cultivation
1. Agrobacterium tumefacienes strain LBA4404 containing pRT99 was grown
overnight in 50 ml of liquid MYB medium (0.5% (w/v) yeast extract,
0.05%(w/v) casein hydrolysate , 0.8% (w/v) mannitol, 0.2% (w/v)
ammonium sulfate, 0.5%(w/v) sodium chloride, pH 6.6). After inoculation,
bacterial cultures were maintained at 28ºC and 120 rev/min in shaking
incubator.
2.
Bacterial cells from strain were then collected by centrifugation at 3,500
rpm for 10 minutes at 4ºC in 15ml Falcon tubes.
3. The pellets were resuspended in a hormone free MS (Murashige and Skoog,
1962) medium.
4. The density of bacterial strain was adjusted at approximately 5×1010
CFU/ml.
5. After one day of preculturing explants were immersed in the bacterial
suspension for 10-15 minutes.
6. Thereafter the explants were blotted on sterilized filter paper and placed on
cocultivation medium containing MS medium with 200µM acetosyringone.
In each plate about 10 explants were cocultivated.
7. These plates with explants were kept in growth chamber at 27 ºC, 16h of
photoperiod, illumination of 45 uEm-² s-¹ and 60% relative humidity, for
two days.
2.2.11: Selection and regeneration
After three days of cocultivation, the explants were washed with washing
medium (WM) that consisted of sterilized MS medium containing 500mg/l
cefotaxime to kill bacteria and blotted on sterilized filter paper. All antibiotics were
filter sterilized and added to the autoclaved medium. The explants were then
transferred to the flasks containing selection medium SM containing MS medium
with 0.1mg/l BAP (Benzyl amino purine) before autoclaving, and 20mg/l Kanamycin,
500mg/l cefotaxime was added with the help of micropipette under the laminar flow
hood, when the autoclaved media cooled down at 45ºC.
These flasks with explants were maintained at 27 ºC, 16h of photoperiod,
illumination of 45 µEm-² s-¹ and 60% relative humidity. The explants were then
transferred to fresh medium (SM) weekly during the first month. Afterwards
subcultures were made every two weeks. After eight weeks, the concentration of
cefotaxime was reduced to 50 mg/L.
2.2.12: Rooting
For rooting, the developed shoots cut off segments of Artemisia dubia were
cultured on Half MS medium containing 0.025mg/L NAA and solidified with 0.1%
gelrite while shoots of Artemisia annua were cultured on MS medium containing
0.1mg/L NAA.
When roots appeared then sub culturing of parent plants were done in order to
increase the number of transformed plants. For sub culturing same medium is used
which was used for rooting containing Half MS medium with 0.025mg/L NAA and
0.1mg/L NAA for Artemisia dubia and Artemisia annua respectively, solidified with
0.1% gelrite for solidification.
2.2.13: Transplantations to pots and acclimatization
After selection of transformed seedlings, the controlled and kanamycin-resistant
plants were transferred individually to small pots containing soil. The soil was made
by mixing equal amounts of clay, sand and peat. Small pores were made in their
bottoms so that seepage of excessive water may occur at the bottom and may not
injure the roots.
Plants after transferring to pots were kept enclosed with transparent polythene
bags to retain moisture. Acclimatization was done for 15 days in growth room
25±2°C, 16h of photoperiod, illumination of 45 uEm-² s-¹ and 60% relative humidity.
Once being hardened, the plants were shifted to green house. Extensive care was
taken till they reached maturity. Number of differences was found in morphological
features of the control and transgenic antibiotic resistant plants. The morphological
parameters were observed and recorded.
2.2.14: Agrobacterium rhizogenes mediated transformation of Artemisia dubia
and Artemisia annua with rol Genes
Agrobacterium rhizogenes strain LBA9402 and LBA8196 were used for the
transformation experiments. A. rhizogenes strains were grown overnight in MYA
medium. Plants were infected with the bacterial strains by the following
transformation method.
2.2.15: In-vitro plant production and sterilization
Transformation with Agrobacterium rhizogenes was carried out with the plants
growing in green house through their in-vitro propagation.
Small stem (2-4cm)
portion with bud of A.dubia and A.annua plants was used. 70% Ethanol (in sterilized
water) for 4 min and solution of commercial bleach (20 ml) with sterilized water (80
ml) and tween 20 (100 µl) was used for 15 min with gentle shaking. It was followed
by two minutes washing with sterile distilled water for 5 times. New plants appeared
from the bud after one month and plantlets were transferred to half MS medium that
were later used for transformation. 24 hours incubated Agrobacterium cultures were
used for infection by making 2-3 slight cuts in stem portion of A. annua and Artemisia
dubia plants with Agrobacterium rinsed scalpel.
2.2.16: Media used for establishment of hairy root cultures
Following media were used for establishment and propagation of continuously
artemisinin producing hairy root cultures.
2.2.16.1: Preparation of MS shooting medium
Shooting medium was prepared by dissolving 4.44 g MS medium
(Murashige and Skoog, 1962) (Cat # M404, Phytotechnology Lab) in distilled water
along with 3 % D-sucrose (Phytotechnology cat # S391), 2.5 μM benzylaminopurine
HCl (Sigma cat # B5920), 0.25 μM naphthaleneacetic acid (Sigma cat # N0640) and
0.5 % g agar gel (Sigma cat # A3301). Distilled water was added to make the final
volume 1 litre. Its pH was adjusted to 5.8 and autoclaved before use.
2.2.16.2: Preparation of ½MS rooting medium
Rooting medium was prepared by dissolving the 2.22 g MS medium (½ MS
medium, Phytotechnology Lab cat # M404) along with 2 % D-Sucrose,
(Phytotechnology Cat # S391) and 0.5 % Agar gel (sigma Cat # A3301) in the
distilled water and final volume was made up to 1 litre. Its pH was adjusted to 5.8 and
autoclaved before use.
2.16.3: Preparation of nutrient agar medium
Nutrient agar medium was prepared by dissolving 23 g Nutrient Agar (Sigma
Cat # N-9405) in distilled water and the volume was made up to 1 litre. Its pH was
adjusted to 7.0 and autoclaved before use.
2.2.16.4: Preparation of B5 solid medium
One litre B5 solid medium was prepared by dissolving its 18 nutrients (detail
of nutrients is in appendix IV) in freshly prepared distilled water along with 3 %
sucrose, 0.25 % phytagel (Sigma cat # P8169) and final volume was made up to 1
litre. Its pH was adjusted to 5.8, autoclaved, poured into petriplates and solidified.
2.2.16.5: Preparation of B5 selection medium
One litre B5 selection medium was prepared by adding 600 mg/L of 0.22 μM
filtered cefotaxime in autoclaved B5 solid medium. Cefotaxime was mixed
thoroughly in B5 solid medium. After mixing, this medium was poured in the
petriplates and solidified.
2.2.16.6: Preparation of B5 liquid medium
One litre B5 solid medium was prepared by dissolving its 18 nutrients in
freshly prepared distilled water along with 3 % sucrose and final volume was made up
to 1 litre. Its pH was adjusted to 5.8, autoclaved and 50 ml media transferred in to 250
ml Erlenmeyer flasks.
2.2.17: Induction of hairy roots
2.2.17.1: Preparation of A. rhizogenes strain
A. rhizogenes strain LBA9402 and LBA 8196 (Kindly provided by Dr.
Fabricio Medina-Bolivar Lab, ABI, and ASU, USA) was used for production of hairy
roots. These Agrobacteria were stored at –70 ºC freezer. Pure cultures of
Agrobacteria were prepared by streaking on the nutrient agar in the petriplates for 24
hours at 25ºC. For inoculation, single colony of this strain was picked and streak
again on the nutrient agar in the petriplates. After inoculation, A.rhizogenes was
placed again in the incubator at 25ºC. After 24 hours, A. rhizogenes was ready for
infection in stem portion of A. Annua and A.dubia.
2.2.17.2: A. rhizogenes infection on Artemisia annua and Artemisia dubia
Twenty eight days old sterile plantlets of A. annua and A.dubia growing in the
magenta box were used for infection and transformation. A single colony of A.
rhizogenes, 24 hours incubated Agrobacterium cultures, was picked with sterile
scalpel which lightly incised into the stem portion of A. annua and Artemisia dubia. In
each plantlet, 2-3 incisions were made on different places along the length of stem, by
Agrobacterium rinsed scalpel. The infected plantlets were incubated again in growth
room having 24 μM m-2 sec-1 cool white fluorescent light and 25ºC with 16/8 light
period. After seven days of infection, hairy roots started emerging from
Agrobacterium infected stem portions of A. annua and A.dubia.
2.2.17.3: Transfer of hairy root cultures on B5 solid medium
These hairy roots were cut from stem portions of both Artemisia species when
they were 1-2cm long and transferred to B5 selection medium in petriplates in such a
way that one root line was transferred in one plate. These petriplates were placed in
dark at 25ºC for two weeks. B5 selection medium was used to kill the excess of A.
rhizogenes from hairy roots. After two weeks, 1-2cm long fast growing root tip was
excised from these root cultures and transferred to B5 solid medium in the petriplates
and incubated again for another two weeks.
2.2.17.4: Transfer of hairy roots in to B5 liquid medium
Ten fast growing root tips emerged from single root line grown in petriplates
were transferred to 50 ml B5 liquid media in 250 ml Erlenmeyer flasks. These flasks
were placed on orbital shaker at 90 rpm in 24 μM m-2 sec-1 cool white fluorescent
light and 25ºC. Hairy roots produced and subcultured after every 2 weeks and
maintained in liquid medium were used for further analysis.
2.2.18: Molecular analysis
For confirmation of the transformation and integration of the desired genes,
molecular analysis was carried out through PCR and Southern blot analysis. Plants
transformed with Agrobacterium tumefacienes and Agrobacterium rhizogenes strains
LBA4404 and LBA8196 respectively were used for PCR and southern blot analysis
for rol A, B and C genes. For PCR and southern blot analysis, genomic DNA was
isolated from transformed plants, and plasmid DNA was also isolated from the strains
of Agrobacterium tumefacienes and Agrobacterium rhizogenes.
2.2.18.1: Isolation of genomic DNA from plant leaves
For extraction of genomic DNA from the seedlings simplified CTAB (Cetyl
trimethyl ammonium bromide) method of Doyle and Doyle, (1990) was used. DNA
was extracted from both transformed and untransformed control plants by grinding
individually frozen seedlings to very fine powder using ice cold pestle and mortar.
5ml CTAB extraction buffer at 65°C was added to the powdered leaves and incubated
at this temperature for 20 minutes with occasional vigorous shaking. 2ml of
chloroform was then added and shake thoroughly and placed on inverter at room
temperature for 20 minutes, then centrifuged at 3000 rpm for 5 minutes to resolve
phases. After which aqueous phase was transferred to fresh tube and 2ml of
isopropanol was added, mixed well and stored on ice for 10 minutes. It was
centrifuged again at 3000 rpm for 5 minutes to collect precipitates. The liquid was
drained away and sides of tube were dried using blotting paper. To dissolve ppt. 200ul
of TE was added. Then 200ul of 4 M LiAc was added and incubated on ice for 20
minutes. It was centrifuged again for 10 minutes at 3000 rpm and to supernatant 1 ml
of absolute ethanol was added in a fresh tube and placed on ice for 20 minutes. To
collect precipitates the tube was centrifuged again for 10 minutes at 3000 rpm. The
liquid was drained away and DNA was dissolved in 200 µl TE by gentle pipeting. 100
µl of 3 M NaOAc was added, and then equal volume of chloroform was added. After
which 2 volume of absolute ethanol was added and stored on ice for 5 minutes to
precipitate DNA. It was then centrifuged for 10 minutes to collect ppt. Ethanol was
evaporated and DNA was dissolved in 200 µl of TE and stored in refrigerator at 20°C.
2.2.18.1.1: COMPOSITION OF CTAB BUFFER
Sorbitol
140 mM
Tris, Ph 8
220 mM
EDTA
22 mM
NaCl
800 mM
Sarkosyl
1%
CTAB
0.8 %
These were combined; pH was adjusted to 8 and autoclaved.
2.2.18.2: Extraction and purification of plasmid DNA
Minipreparations of plasmid DNA were obtained by alkaline lyses method
described below.
Agrobacterium strains were grown overnight in 50ml of liquid MYB medium.
Medium was supplemented with 50 mg/L kanamycin sulphate, added to the
cold media, after autoclaving under the laminar flow hood. After inoculation,
bacterial culture was maintained at 28°C and 120 rev/min in shaking
incubator.
1.5 ml of bacterial culture was poured into four microfuge tubes and
centrifuged at 14,000 rpm for 15 minutes at 4°C to collect the cells. The
supernatant was removed by micropipette and the cells were left to dry.
The pellet was resuspended in 100 µl of solution 1 by vortexing and was left at
room temperature for 10 minutes.
200 µl of solution 2 was added and the contents were mixed by inverting the
tubes for 4-5 minutes and left at room temperature for 5 minutes.
Then 100 µl of solution 3 was added and the contents were mixed gently for 45 times, the tubes were stored on ice for 20-30 minutes. Low temperature and
pH precipitates the denatured DNA which was pelleted by centrifugation at
14,000 rpm for 10 minutes and the supernatant was transferred into fresh tube.
To purify DNA equal volume of chloroform was added and centrifuged again
for 5 minutes at 14,000 rpm. The supernatant was transferred to a fresh tube
and double volume of ethanol was added to supernatant and stored at -20°C
for 1 hour to precipitate DNA and centrifuged for 10 minutes at 14,000 rpm to
collect the ppt.
The pellet was washed twice with 70% ethanol to remove any remaining salt.
After drying, the pellet was resuspended in 30 µl of TE buffer and was then
stored at -20°C in refrigerator.
2.2.18.2.1: SOLUTIONS
Solution 1
Solution 2
Glucose
50 mM
Tris
25 mM (pH 8.0)
EDTA
10 mM (pH 8.0)
SDS
1%
NaOH
0.2 N
Solution 3
Sodium Acetate
3M
TE
Tris pH 8.0
10 mM
EDTA
1 mM
2.2.19: Polymerase chain reaction
Polymerase chain reaction for the detection of rol A, B and C gene was
performed following the standard method of Taylor, (1991). PCR reaction was
performed in 0.2 ml tubes containing 50 µl total reaction mixture. DNA (0.3 µg) was
incubated in a final volume of 50 µl with 0.25 µg of reverse and forward primers, 0.2
mM each of d ATP, d GTP, d CTP, and d TTP and 2 mM MgCl2 and 2 units of Taq
polymerase and 5 µl of 10X PCR buffer. The reaction mixture was centrifuged for
few seconds thorough mixing.
The reaction mixture was taken through thermo cycling conditions as: 5
minutes of 95°C for template denaturation followed by 25 cycles of amplification
each consisting of 3 steps; 30 seconds at 95°C for DNA denaturation into single
strands; one minute at 53-55°C for rol A, 55-57°C for rol B and 54-56°C for rol C
primers to hybridize or ―anneal‖ to their complementary sequences on either side of
the target sequence; and one minute at 70°C for extension of complementary DNA
strand from each primer. Final 10 minutes at 70°C for Taq polymerase to synthesize
any unextended strands left. PCR was performed using gene Amp PCR system 2400
and gene Amp PCR system 9600 thermocyclers (Perkin Elmer, USA).
2.2.19.1: Primers used during PCR
During PCR following forward and reverse primers for rol A, B and C gene were
used.
1. rol A gene forward 5‘-AGAATGGAATTAGCCG GACTA-3‘, reverse primer
5‘-GTATTAATCCCGTAGGTTTGTT-3‘,
2. rol B forward primer 5‘-GCTCTTGCAGTGCTAGATTT-3‘, reverse primer
5‘-GAAGGTGC AAGCTACCT CTC-3‘,
3. rol C gene forward 5‘-GAAGACGACCTGTGTTCTC-3‘, reverse primer 5‘CGTTCAAACGTTAGCCGA TT-3‘.
2.2.19.2: Agarose gel electrophoresis
Agarose gel electrophoresis was carried out to analyze the amplified DNA
samples. 1.5 percent w/v agarose gel was prepared by melting 1.5 gm of agarose in
100 ml of 1 X TBE buffer in microwave oven. 4 µl (4 µl/ 100 ml) gel red solution was
added to stain DNA. DNA samples were mixed with DNA loading dye containing
0.25% bromophenol blue prepared in 40% sucrose solution. Electrophoresis was
performed at 100 volts (50 mA) for 65 minutes in 1 X TBE running buffer. After
electrophoresis amplified product was detected by placing the gel on UV-Trans
illuminator (Life Technology, USA).
COMPOSITION OF 10 X TBE
Tris
890 mM
Boric acid
25 mM
EDTA
0.1 mM (pH 8.3)
2.2.20: Southern blot analysis
Southern blot analysis of transformants was performed by extracting the
genomic DNA from the plant leaves by using CTAB (Cetyl trimethyl ammonium
bromide) method of Doyle and Doyle, (1990). The DNA was digested and agarose gel
electrophoresis was carried out and the separated DNA fragments were transferred to
a positively charged nylon membrane. The probe was prepared and hybridization of
membrane carrying plant DNA was done with the probe. Finally the membrane was
placed over an x-ray film for exposure.
2.2.20.1: DNA restriction
Digestion restriction was carried out by using restriction endonuclease under
conditions recommended by the manufacturer (Promega). Approximately 50 µg of
genomic DNA isolated from both transformed and untransformed plants were
digested with EcoRI in 10 μl reaction:
DNA
50 µg
KPNI
(1 U/μl) 0.2 μl
10X Buffer
1 μl
PCR H2O
to 10 μl
The digestion mixture was incubated at 37 °C for 2 hours in 10 μl reaction. The DNA
was subsequently precipitated with ethanol and sodium acetate, resuspended in 15 μl
TE buffer after washing with 70% ethanol.
2.2.20.2: Agarose gel electrophoresis
DNA loading buffer (5 μl) was added to each sample and separation of
digested DNA was carried on 0.8% (w/v) TBE agarose gel containing 0.5 μg/ml (w/v)
ethidium bromide. Electrophoresis was carried out for 16 hours at 40mA constant
current.
2.2.20.3: Transfer of restriction fragments to membrane
The agarose gel containing the separated DNA fragments was treated for 15
min. in 0.25 M HCl at RT, and then shaken for 30 min. in denaturing solution
(Appendix-V) and for another 30 min. in neutralizing solution (Appendix-V). The
DNA fragments were transferred from the gel to the Hybond-N+ nitrocellulose
membrane overnight with 20 X SSC buffer. For fixation of the DNA fragments, the
membrane was exposed to UV light for 5 min. and baked at 80°C for 2 h.
2.2.20.4: Labeling of DNA
PCR products of rol A gene from plasmid was used as the probe. The probe
was labeled using digoxigenin (DIG)-11-dUTP with DIG High Prime DNA Labeling
reagents (Roche, Mannheim, Germany). 15 μl probe was added in 25μl sterile
distilled water and 10 μl hexanucleotide in a total volume of 50 μl and incubated for 5
min. at 95°C for denaturation. Then it was quickly chilled on ice. Finally, 3 μl mix i.e.
2 μl (DIG)-11-dUTP and 1 μl Klenow enzyme (5 U/μl), were added and incubated for
10 min. at 37°C. After incubation, 4 μl dNTPs was added and it was reincubated for 5
min. at 37°C. The reaction was stopped with 50 μl TE buffer; pH 8 and the probe was
allowed to pass through a sephadex column (to clean the probe). Before using the
probe, it was incubated at 95°C for 5 min. for denaturation and quickly chilled on ice.
2.2.20.5: Hybridization process
The pre-hybridization was performed at 45°C in 50-100 ml hybridization
solution (Appendix-V) without adding labeled DNA. After 3 hours the hybridization
solution was discarded and replaced with the fresh solution, after adding labeled DNA
probe, hybridization was performed overnight at 45°C. Washing of the membrane was
carried out thrice with 50 ml washing buffer (20X SSPE, 10% SDS and dH2O 408ml)
at 65°C for 20 minutes. Finally, Kodak hyper-film (X-ray) was exposed with the
hybridized membrane for 3-5 days at -70°C.
2.3: Results
The aim of the present study was to optimize the transformation conditions
for A.annua and A.dubia, establishment of hairy root cultures and production of
different transgenic lines of both species. Molecular analysis was performed for
confirmation of transformation through PCR and Southern blot analysis.
Agrobacterium tumefaciens strain LBA4404 containing pRT99 harboring rol genes
and Agrobacterium rhizogenes strain LBA8196 and 9402 were used for the
transformation experiments. Numbers of factors have been reported that affect the
efficiency of transformation with A.tumefaciens and A.rhizogenes, different
parameters like seed surface sterilization with different time durations, effect of type
of explants and co-cultivation period were studied.
2.3.1: Agrobacterium tumefaciens mediated transformation of Artemisia dubia
and Artemisia annua with rol ABC Genes
Agrobacterium tumefacienes strain LBA4404 containing pRT99 harboring rol
ABC genes were used for the transformation experiments. The A. tumefacienes strain
LBA4404 grown overnight in MYA medium. Explants were infected with the
bacterial strains following the transformation method described in material and
methods.
2.3.2: Seed surface sterilization
Seeds of both Artemisia species were surface sterilized with Mercuric chloride
(HgCl2) 0.1% (w/v) followed by three times washing with sterilized distilled water.
Seeds were exposed to sterilizing agents for varying durations. No contamination was
observed in seeds of all treatments. The number of seeds germinated varied with
different duration of exposure to the sterilizing agent i.e. Mercuric chloride (HgCl2)
0.1% (w/v). The germinated seeds are shown in figure 2.2 and 2.3.
2.3.3: Medium for seed germination
Seeds sown on half strength MS medium showed 100% germination within 3
days. Whereas on plain agar although 80% of the seeds germinated but the duration
was almost 6 days (as shown in the table 2.1). Moreover, the seeds germinated on half
MS medium were healthier and fresh as compared to the ones on plain agar.
Table 2.1: Effect of medium on germination of seeds.
Medium
Duration of germination
Percentage of seeds germinating
½ MS
3 days
100%
Plain agar
6 days
80%
120
Percentage of seed germination
% age seed germination
100
80
60
40
20
0
2 min
5 min
10 min
Sterilizartion time
20 min
Fig 2.2: Percentage seed germination with different duration of exposure
to 0.1% (w/v) Mercuric Chloride
(a)
(c)
(b)
(d)
Fig 2.3: In vitro grown seedlings of Artemisia dubia (a, b)
and Artemisia annua (c, d) on MS.
2.3.4: Types of explants
The Agrobacterium tumefaciens mediated transformation procedure
involving tissue culturing was carried out with two different types of explants i.e.
stem and leaf with one strain of Agrobacterium tumefaciens i.e. LBA4404 containing
pRT99 harboring rol ABC genes. Explants were prepared by cutting leaf and stem
segments from 20 days old seedlings (as shown in figure 2.4) into 0.5-1 cm pieces and
transferred to MS medium containing 200µM acetosyringone. These pieces were used
as explants for further manipulation.
(a)
(b)
(c)
(d)
Fig 2.4: Leaf and stem explants used for transformation Artemisia dubia (a, b)
and Artemisia annua (c, d)
2.3.5: TRANSFORMATION
2.3.5.1: Effect of co-cultivation period on transformation
Explants from 20 days old in vitro grown seedlings (Fig. 2.4) were pre-cultured
for three days on co-cultivation medium containing MS with 200µM acetosyringone.
After pre-culturing, explants were co-cultivated with bacterial culture and were put on
the co-cultivation medium. After two days on co-cultivation medium, explants were
shifted to the selection regeneration media. Leaf and stem explants were incubated for
3, 5, 10 and 15 minutes in Agrobacterium solution as shown in fig 2.5 a (A.dubia) and
2.5 b (A.annua). Highest (70%) regeneration response was observed in explants
incubated for 5 minutes. Further increase in the length of incubation decreased the
transformation frequency as shown in figure 2.6.
2.3.5.2: REGENERATION
2.3.5.2.1: Effect of antibiotics on Agrobacterium in regeneration medium
To control Agrobacterium growth in selective regeneration medium after
transformation cefotaxime 500 mg/L was used. Cefotaxime (Claforan®, Hoechst AG,
and Frankfurt, Germany) shows good antimicrobial activity against Agrobacteria
strains and exhibits an auxin like activity on plant material Vergauwe et al. (1996b).
In some experiments Agrobacterium growth occurred again when regenerated
transformed shoots were transferred to fresh regeneration medium without cefotaxime
after 4 weeks and these regenerated shoots died due to over growth of Agrobacterium.
Therefore, in other experiments cefotaxime was used for two months after their
transfer to regeneration medium
2.3.5.2.2: Regeneration of transgenic plants
Leaf and stem explants excised from one week old seedlings were cocultured
with Agrobacterium on cocultivation medium. After three days these explants were
washed with sterile distilled water and then with cefotaxime to remove excess of
Agrobacterium. Leaf apices were cultured for direct shooting on SRM 2 containing
0.1mg/L BAP supplied with Kanamycin 20mg/L and cefotaxime 500mg/L. After two
weeks transgenic shoots were obtained (Fig. 2.7).
(a)
(c)
(b)
(d)
Fig 2.5 (a): Regeneration response of Artemisia dubia explants on selection
medium after incubation in Agrobacterium solution for various durations.
(a). 3 min
(b). 5 min
(c). 10 min
(d). 15 min
(a)
(c)
(b)
(d)
Fig 2.5 (b): Regeneration response of Artemisia annua explants on selection
medium after incubation in Agrobacterium solution for various durations.
(a). 3 min
(b). 5 min
(c). 10 min
(d). 15 min
Fig 2.6: Effect of incubation period in Agrobacterium solution
on regeneration efficiency of explants.
(a)
(b)
©
(d)
Fig. 2.7: Shooting of transformants from different explants
Artemisia dubia
(a) Leaves
(b) Stem
Artemisia annua
(c) Leaves
(d) Stem
2.3.5.3: Rooting of transformed plants
For rooting, when the transgenic shoots were elongated to 2-4cm, the
developed shoots of Artemisia dubia cut off segments were cultured on rooting
medium (RM) i.e. half strength MS (Murashige and Skoog, 1962) medium containing
0.025 mg/L NAA while developed shoots of Artemisia annua were cultured on
rooting medium (RM) i.e. full strength MS (Murashige and Skoog, 1962) medium
containing 0.1 mg/L NAA. Rooting was quantified on the basis of percentage of
shoots showing response for rooting. The rooting response in transformed plants was
found to be 50% and has been shown in figure 2.8.
(a)
(b)
©
(d)
Fig 2.8: Rooting response of transformed plants
Artemisia dubia (a, b) and Artemisia annua (c, d)
2.3.6: Morphological analysis of transformed plants
Clear morphological differences were found in some of the green house grown
transgenic plants as compared to control plants of both Artemisia species. The
morphological differences studied had been listed in table 2.2 and represented in
figure 2.9. Control plants were healthier and survived longer as compared to
transgenic plants. Marked differences exist in height between control and transgenic
plants growing in a greenhouse.
Table 2.2: Morphological differences observed among Transgenic and Control
Plants
Morphological
Control Plants (cm) + S.E
characters
Average
Transgenic Plants (cm)
+ S.E
Plant 87cm + 0.5
70cm + 0.3
height
Stem
Straight, unbranched and soft in Branched and hard in
texture
texture
Leaves
Large size and broad
Small size and narrow
Inflorescence
Axial, without hairs
Terminal, excessive hairy
(a)
(b)
(c)
(d)
Fig. 2.9: Comparison of transformed and non-transformed plants
(a) Non-Transformed plant of A. dubia
(b) Transformed plant of A. dubia
(c) Non-Transformed plant of A. annua
(d) Transformed plant of A. annua
Agrobacterium rhizogenes mediated transformation of Artemisia dubia and
Artemisia annua with rol genes
Agrobacterium rhizogenes strain LBA9402 and LBA8196 rol genes were used
for the transformation experiments. The A. rhizogenes strains grown overnight in
MYA medium. Plants were infected with the bacterial strains following the
transformation method described in material and methods.
2.3.7: In-vitro plant production and sterilization
For Agrobacterium rhizogenes mediated transformation, small stem explants
carrying a bud were excised from green house grown plants of both Artemisia species
and cultured on MS medium. New plants appeared from bud after one month; these
plantlets were cut and then transferred to half MS medium (Fig 2.10). The
subculturing of these plantlets to half MS medium resulted in the propagation of
multiple plantlets from a single bud.
(a)
(b)
Fig. 2.10: In-vitro grown plantlet of (a) Artemisia dubia (b) Artemisia annua
2.3.8: Production of hairy roots
The four-week old plants of both Artemisia species from half MS medium were
infected with Agrobacterium rhizogenes strains LBA9402 and LBA8196; hairy roots
appeared after six days of infection (Fig. 2.11), whereas no hairy roots were produced
with the strain LBA8196.
2.3.9: Molecular analysis of transformed plants
Molecular analysis of the seedlings transformed by rol ABC genes, obtained
from tissue culturing was performed.
2.3.9.1: Genomic DNA extraction
For PCR analysis, genomic DNA was extracted using CTAB method (Cetyl
trimethyl ammonium bromide) of Doyle and Doyle, (1990) from the leaves of the
transformed plants. DNA extracted through this procedure was run through the Gel (1%
agarose) and visualized under U.V transilluminator.
(a)
(b)
Hairy Roots
©
(d)
Fig. 2.11: Induction of hairy roots from stems infected with Agrobacterium
rhizogenes.
(a, b) A. dubia (c, d) A. annua
2.3.9.2: Polymerase chain reaction (PCR)
For molecular analysis, genomic DNA was isolated by using CTAB method of
Doyle and Doyle, (1990) from transformed and untransformed plants of both Artemisia
species, and plasmid DNA was also isolated by using alkaline lysis method from both
Agrobacterium tumefaciens and Agrobacterium rhizogenes. PCR was performed for rol
A, B and C genes and the amplified products (308bp, 779bp, 540bp respectively) were
observed to confirm transformation. Same size amplified product was also obtained
from the plasmid DNA of Agrobacterium strains. Rol A, B and C genes were detected in
all transformed lines of Artemisia dubia (D1, D2, D3, DH1 and DH2) and Artemisia
annua (A1, A2, A3, AH1 and AH2) as well as in plasmid DNA but not in control plants
(Fig. 2.12a and 2.12b). The figure 2.12 shows the amplified products of rol A, B and C
genes.
M
P
D1
D2
D3
DH1
DH2
C
3000
2000
1000
308bp
750
500
250
(a)
M
C
P
D1
D2
D3
DH1
DH2
3000
2000
1000
750
500
250
779bp
(b)
M
C
P
D1
D2
D3
DH1
DH2
3000
2000
1000
750
540bp
500
(c)
250
Fig. : 2.12 (A). PCR analysis showing amplified product of (a) rol A
(b) rol B and (c) rol C in transgenic Artemisia dubia
3000
M
C
P
A1
A2
A3
AH1
AH2
2000
1000
750
500
308bp
250
(a)
M
C
AI
A2
A3
AH1
AH2
P
3000
2000
1000
750
779bp
500
250
(b)
M
C
AI
A2
A3
AH1
AH2
P
3000
2000
1000
750
540bp
500
(c)
250
Fig. : 2.12 (B). PCR analysis showing amplified product of (a) rol A
(b) rol B and (c) rol C in transgenic Artemisia annua
Fig 2.12: PCR analysis showing amplified products (A) D1-D3 represents the plants
transformed by Agrobacterium tumefaciens and DH1-DH2 represents the plants
transformed by Agrobacterium rhizogenes and in Artemisia annua (B) A1-A3
represents the plants transformed by Agrobacterium tumefaciens and AH1-AH2
represents the plants transformed by Agrobacterium rhizogenes. Lane P represents the
plasmid DNA. Lane C refers to the non transformed control plants. Lane M
corresponds to 1 kbp Ladder (Fermentas).
2.3.10: Southern blotting
Southern blot analysis of PCR-positive plants of Artemisia dubia and Artemisia
annua was performed by DIG High Prime DNA Labeling and Detection Starter Kit II
(Roche Cat. No. 11585614910) according to the manufacturer‘s instructions.
Hybridization bands were detected in Southern blots with the rol A probe in the
transgenic plants. In all the transformants, the inserted copy number was one except line
D2 of Artemisia dubia and A1 and A3 of Artemisia annua in which two copies of the
inserted genes were observed (Fig. 2.13 a,b). The results confirmed the integration of
the Agrobacterium T-DNA in the genome of Artemisia dubia and Artemisia annua
transgenic lines.
C
D1
D2 D3 DR1 DR2
4.5 kb
4.0 kb
3.5 kb
3.0 kb
2.5 kb
2.0 kb
(a)
C
A1
A2
A3 AH1 AH2
4.5 kb
4.0 kb
3.5 kb
3.0 kb
2.5 kb
2.0 kb
(b)
Fig. 2.13: Southern blot analysis of (a) Artemisia dubia (b) Artemisia annua. C
untransformed control plant, lanes 1–5 rol ABC transformed plants (Artemisia dubia),
lanes 1-5 rol ABC transformed plants (Artemisia annua), D1-D3 represents the plants
transformed by Agrobacterium tumefaciens and DH1-DH2 represents the plants
transformed by Agrobacterium rhizogenes (Artemisia dubia), A1-A3 represents the
plants transformed by Agrobacterium tumefaciens and AH1-AH2 represents the
plants transformed by Agrobacterium rhizogenes (Artemisia annua).
2.3.11: Acclimatization
Transformed rooted plantlets of both Artemisia species were shifted to small
pots containing a mixture of soil and peat moss. Plants were then acclimatized in
larger pots in growth chamber at 25 ±2ºC, 16 h of photoperiod, illumination of 45
uEm-² s-¹ and 60%
and 60 % relative humidity before transfer to the harsher conditions of the green
house environment. Out of 15 plants shifted to pots, only 8-10 seedlings were able to
survive the growth room conditions, so the percentage of seedlings acclimatized to the
outer environment was 53-66%. Rest of the seedlings could not withstand in the
growth room (Fig: 2.14a, b).
(a)
(b)
©
(d)
Fig. 2.14: Transformed plant in green house
(a, b) Transformed plant of A. dubia
(c, d) Transformed plant of A. annua
2.4: Conclusion
In this part, transformation of Artemisia annua and Artemisia dubia with rol
genes was carried out through A.tumefacienes and A.rhizogenes and following
conclusion were drawn:
Both species of Artemisia (A.annua and A.dubia) have shown variable
regeneration responses and both proved best for in vitro regeneration.
Factors like genotype, explants type, cocultivation period and
sterilization time affect the in vitro regeneration ability.
Among the two explants (Stem and Leaf) used for in vitro
regeneration, maximum shoot formation was observed in stem explants
followed by leaf explants.
Maximum shoot regeneration was recorded on MS + 0.1 mg/l BAP +
20mg/ml kanamycin + 500mg/l cefotaxime.
Rooting was observed on half MS medium with 0.025mg/l for A.dubia
while half MS medium with 0.1mg/l was used for A.annua shoots.
Agrobacterium mediated transformation was the preferred method of
Artemisia transformation dependant on optimum combination of
sterilization time, inoculation time and co-cultivation period for stem
and leaf explants.
Stable transformation of Artemisia with rol genes was carried out
successfully
by
employing
Agrobacterium
tumefacienes
and
Agrobacterium rhizogenes mediated transformation.
Transformation
and
insertion
of
desired
genes
from
both
transformation methods was confirmed by PCR and southern blotting.
Chapter No. 3
Analysis of Artemisinin and its Derivatives and their comparison in transformed and untransformed plants of Artemisia annua and Artemisia dubia
3.1: Introduction
Artemisinin an endoperoxide sesquiterpene lactone produced by aerial parts of
Artemisia plant is effective even against multi-drug resistant strains of the malarial
parasite. The isolation and characterization of artemisinin from Artemisia plant is
considered as one of the most novel discoveries in recent medicinal plant research
(Roth and Acton, 1989). Although the complete organic synthesis has been
established, but chemical synthesis of artemisinin is not yet economically feasible
because of its complexity and low yield. Currently the leaves, roots and flowers of
Artemisia species form the only source of this drug. Artemisinin is found in very low
quantities (0.05%-1.1%) in different cultivars of Artemisia dubia and Artemisia annua
(1%-4%). High artemisinin yielding clones are being isolated by selection and other
non-conventional approaches; however, these have their own limitations (Delabays et
al., 1993). Genetic improvement of natural varieties has been attempted but the
maximum yield of artemisinin from this route reached so far is 2% (Graham et al.,
2010). Through transformation we can enhance the production of artemisinin in
Artemisia dubia and Artemisia annua by inserting our desired genes.
A number of publications have reported large differences in artemisinin content
depending on variety, season, different plant parts, co cultivation conditions and plant
developmental stage (Lommen et al., 2007; Davies et al., 2009; Yang et al., 2009).
Artemisinin has been detected in leaves; small green stems, buds, flowers and seeds
(Martineg and Staba, 1988; Ferreira et al., 1995, Abdin et al., 2003). Reports on the
distribution of artemisinin throughout the plant are however inconsistent. Many
analytical procedures to identify and quantify artemisinin, its biosynthetic precursors
as well as its metabolites have been developed during the last 30 years. These include
thin layer chromatography (Klayman et al., 1984), High performance liquid
chromatography with UV detection (Zhao and Zeng, 1985), gas chromatography
coupled to mass spectrometry (Woerdenbag et al., 1991), capillary electrophoresis
coupled to UV detector (Christen and Veuthey, 2001), HPLC with evaporative light
scattering detection (Kohler et al., 1997; Christen and Veuthey, 2001) and enzymelinked immunosorbent assay (Jaziri et al., 1993).
The aim of the work presented in this chapter was to analyze and compare
artemisinin and its derivatives (artemether, arteether, dihydroartemisinin and
artesunate) in different tissues (transformed shoots, transformed roots and hairy roots)
of transformed and untransformed plants of Artemisia annua and Artemisia dubia.
3.2: Materials and Methods
3.2.1: Extraction of artemisinin and derivatives of artemisinin
Artemisinin and derivatives of artemisinin were extracted from transformed
and non-transformed shoots, roots and hairy roots of Artemisia dubia and Artemisia
annua by using following method:
Biomass extraction was performed according to the method by Lapkin et al.
(2006). Basically, 1 g of plant leaves was extracted with 30ml hexane/ethyl acetate
(9.5:0.5) at room temperature in a Sonication bath (Kerry Ultrasonic UK) for 1 hour.
Hexane and ethyl acetate mixture was used to increase the solubility of artemisinin in
the extraction solvent. The extract was filtered using Whatman 1.0um filter paper and
concentrated in vaco to give the crude extract (Solvent 1).
For quantitative analysis of artemisinin and its derivatives, an aliquoted volume
of the crude extracts were dried in a speed vacuum (Jousan RC1022) at room
temperature and re-suspended in acetonitrile to dissolve artemisinin and other
acetonitrile soluble components of the extract while precipitating waxes and other
non-soluble components (Solvent 2). The suspension was filtered with a syringe filter
(0.45um Millipore) into an HPLC vial ready for analysis.
3.2.2: Reagents used
HPLC grade hexane, ethyl acetate and acetonitrile were obtained from Fisher
scientific UK. Artemisinin, artemether, arteether, artesunate and dihydroartemisinin
reference standards (>98.0 %) were obtained from Sigma-Aldrich (UK). Deionised
water was obtained from Millipore Q-POD (UK) purification unit.
3.2.2.1: Solvents used
3.2.2.1.1: Solvent No.1: Mixture of ethyl acetate and hexane in the ratio of 5:95
For 10 ml we have to add 0.5ml Ethyl acetate and 9.5ml Hexane
For 500 ml we have to add 25 ml Ethyl acetate and 475 ml Hexane
3.2.2.1.2: Solvent No.2: Mixture of acetonitrile and water in the ratio of 50:50
For 10 ml we have to add 5ml Acetonitrile and 5ml Water
For 500 ml we have to add 250 ml Acetonitrile and 250 ml Water
Also we can use 100% Acetonitrile
3.2.2.1.3: Biomass and solvent ratio
Biomass and solvent ratio should be 1:10
For 1g plant material we should add 10ml of solvent
3.2.3: Analysis of artemisinin content
Artemisinin and its derivatives were extracted from shoots, roots and hairy
roots of transformed and untransformed plants of Artemisia dubia and Artemisia
annua and HPLC analysis of extracts was performed on a Shimadzu Prominence
HPLC-UV/ELSD instrument equipped with auto-sampler, degasser and a photo-diode
array detector. The protocol employed was adapted after Lapkin et al. (2006).
Separation was achieved on a Zorbax Eclipse XDB column (15 x 4.6cm, 0.5m)
using a 50% acetonitrile mobile phase flowing at 1ml/min and Injection volume of 20
µl. Detection and quantification of artemisinin was carried out at 210nm using
calibrated standard.
3.2.3.1: High performance liquid chromatography
Chromatographic analysis of plant extracts was performed on a Shimadzu
Prominence HPLC 20A, equipped with auto-sampler, degasser, photodiode array and
evaporative light scattering (ELSD) detector. The protocol employed was modified
after Lapkin et al. (2009). The separation and identification of artemisinin and its
derivatives in extracts were achieved by a 50:50 acetonitrile/water mobile phase
flowing at 1ml/min on an Agilent Eclipse Zorbax column (15 x4.6cm, 0.5um). Due to
absorption at low UV wavelength of artemisinin and its derivatives, ELSD was used
in series with UV for the detection and quantification. Using this protocol, the
artemisinin, artemether, arteether, artesunate and dihydroartemisinin peaks were well
resolved therefore there was no need for a buffered mobile phase to aid separation.
Identification of peaks in extracts was analyzed by comparison with retention indices
of reference standards and by spiking of extracts with appropriate standards.
Before the injection of samples and standards the HPLC system was allowed to
pump the mobile phase and to equilibrate for 1hour until a flat consistent baseline was
achieved. For safe use of HPLC system, every day, after last sample run, mobile
phase was run again followed by run of 50 % and 100 % methanol for 30 minutes
each. The mobile phase used was at pH 7.0, because a higher pH can dissolve the
column.
3.2.3.1.1: Preparation of mobile phase
Mobile phase was prepared by adding together 50:50 acetonitrile/water and
filtered with 0.22 μm aqueous filter (cellulose acetate membrane, Sartorius).
Furthermore, mobile phase was sonicated before use for 40-45 minutes to remove the
air that may interrupt the normal operations of HPLC as well as results of samples.
3.2.3.1.2: Preparation of dilutions of standard
3.5mg/ml
of
artemisinin,
artemether,
arteether,
artesunate
and
dihydroartemisinin reference standards were prepared in acetonitrile from which a
serial dilution of the standards were made to give the following concentrations of
1.75, 0.70, 0.35, 0.18 and 0.04 mg/ml (or 12.21, 6.21, 2.48, 1.24, 0.62 and 0.12 M)
of artemisinin. HPLC analysis of the dilutions produced a calibration graph from
which the qualitative and quantitative analysis of artemisinin and its derivatives in
crude extracts were determined.
3.2.3.1.3: Column used for HPLC
Samples were analyzed by HPLC using Eclipse Zorbax SB C18 column (150 x
4.6mm x 5μm) of Agilent technologies.
3.2.3.1.4: Flow of mobile phase through HPLC system
Flow of mobile phase through column (stationary phase) was 1 ml/min.
3.2.3.1.5: Detector
Diode Array Detector (G1315B-DAD) showed maximum absorbance of
artemisinin and its derivatives at 260 nm.
3.2.3.1.6: Retention time of peak
Retention / elution time peak of artemisinin and derivatives was 40 minute.
Artemisinin and its derivatives were identified by using the authentic standard of
artemisinin and its derivatives.
3.2.3.1.7: Injection volume
An injection volume of 20 μl of sample as well as standard was used.
3.2.3.1.8: Calibration curve
Six dilutions1.75, 0.70, 0.35, 0.18 and 0.04 mg/ml (or 12.21, 6.21, 2.48, 1.24,
0.62 and 0.12 M) of standard artemisinin, artemether, arteether, artesunate and
dihydroartemisinin were used. The calibration curves were obtained by plotting the
chromatographic peak area (mAU) against the concentration (g/ml), where a linear
response was observed. Linear regression analysis resulted in the equation with an
excellent correlation coefficient i.e. R2 = 1. The calibration curve and linear
regression equation were used for the determination of artemisinin in the samples
(Guo et al., 2005). The linear regression equation according to standard
concentrations (g/mL) is as under:
y = mx + b
m = 1.69159 e–1
b = 7.20139e–1
y = 1.69159 e–1x + 7.20139e–1
Where; y = values [Peak Area (mAU)] on y-axis
x = values [Concentration (g/ml)] on x-axis
3.2.3.1.9: Quantification of artemisinin and its derivatives in sample
For any given Peak Area (mAU) i.e. y from sample chromatogram,
corresponding concentration (g/mL) of sample can be calculated by evaluating x as
follows:
x = (y - 7.20139e–1) / 1.69159 e–1.
3.2.3.1.10:
Verification
of
artemisinin
and
derivatives
of
artemisinin
concentrations
Co-injection was used in order to positively identify peaks as artemisinin,
artemether, arteether, artesunate and dihydroartemisinin. Suspected artemisinin and its
derivatives peaks were noted, and their area recorded from the original sample that
was run. An aliquot of a concentrated, Q260 derivatized, filtered (0.22 µm FP-200
13mm
FPVericel™
membrane
filter,
Pall-Gelman
Laboratory)
artemisinin,
artemether, arteether, artesunate and dihydroartemisinin standard (Sigma-Aldrich, St.
Louis, MO) solutions were added to the analyzed sample to achieve an artemisinin
and derivatives peak area about four times that of the area of the putative artemisinin
and derivatives peak. If the putative peak increased in area to an amount that was 4-5
times its original area, the peak was determined to be that peak respective to its
standard. Quantitation was based on the original injection prior to the addition of the
standard.
3.2.4: Statistical analyses
All experiments were done at least in triplicate for each tissue, using an
average artemisinin concentration of three plants of each species. Data were
statistically analyzed using ANOVA and Duncan‘s Multiple Range Test.
3.3: Results
3.3.1: Analysis of artemisinin content in transformed and untransformed
Artemisia annua and Artemisia dubia
Artemisinin was analyzed in plants of Artemisia dubia and Artemisia annua.
Artemisinin was extracted from shoots, roots and hairy roots by using method
described in materials and methods (Section: 3.2.1). Artemisinin was detected on
HPLC at 210nm and retention time of 10.17 in case of A.annua plants (Fig. 3.1) and
9.24 in case of Artemisia dubia (Fig 3.2).
The average artemisinin content from three transgenic lines of Artemisia
annua varied between tissues, the difference between the lines is shown in fig. 3.3. In
the shoots this was 46.89 mg/g dry weight (DW) compared to 4.37 mg/g DW in the
controls shoots. Roots of transformed plants had an average artemisinin content of
3.06 mg/g DW while the amount found in transformed hairy roots was significantly
greater (15.37 mg/g DW) compared to the control roots in which negligible amounts
could be detected (0.02 mg/g DW). The average artemisinin in transgenic lines of A.
dubia was lower than A. annua this was 3.78 mg/g DW in shoots compared to 0.11
mg/g DW in control shoots. The transformed plant roots showed 1.17 mg/g DW and
hairy roots 1.62 mg/g DW of A.dubia, whereas no artemisinin was found in control
roots of A. dubia (Fig. 3.3).
In general, higher amount of artemisinin was observed in transgenic plants of
Artemisia annua as compared to transgenic plants of Artemisia dubia (Fig. 3.3).
(a)
(b)
(c)
Fig. 3.1: HPLC Chromatograms of (a) Standard Artemisinin (b) Artemisia annua
Sample (A2) (c) Spike Sample (A.annua sample + standard Artemisinin)
(a)
(b)
(c)
Fig. 3.2: HPLC Chromatograms of (a) Standard Artemisinin (b) Artemisia dubia
Sample (D2) (c) Spike Sample (A.dubia sample + standard Artemisinin)
60
Artemisinin content (mg/g) DW
50
40
30
20
10
0
Fig. 3.3: Comparative analysis of Artemisinin content in shoots, roots and hairy
roots of transformed and un-transformed plants of Artemisia dubia and Artemisia
annua. The average of three plants is shown together with error bars showing
SE. A2+Artemisinin and D2+ Artemisinin are the spike samples used for
verification of artemisinin concentration
3.3.2: Analysis of derivatives of artemisinin (Artemether, Arteether, Artesunate
and Dihydro-artemisinin) in transformed and untransformed Artemisia annua
and Artemisia dubia plants
Artemether, arteether, artesunate and dihydro-artemisinin were analyzed from
plants of Artemisia dubia and Artemisia annua. These were extracted from shoots,
roots and hairy roots by using method described in materials and methods (Section:
2.23). Artemether, arteether, dihydroartemisinin and artesunate were detected on
HPLC at 210nm and retention time of 7.57, 2.85, 4.79, and 1.94 respectively in case
of A.annua plants (Fig. 3.4, 3.6, 3.8, 3.10) and 7.51, 2.88, 4.97, 1.84 respectively in
case of Artemisia dubia (Fig. 3.5, 3.7, 3.9, 3.11).
The average artemether content from three transgenic lines of Artemisia annua
varied between tissues, the difference between the lines is shown fig. 3.12. In the
shoots this was 4.3 mg/g dry weight (DW) compared to 1.05 mg/g DW in the controls
shoots. Roots of transformed plants had an average artemether content of 1.5 mg/g
DW while the amount found in transformed hairy roots was significantly greater (4
mg/g DW) compared to the control roots in which negligible amounts could be
detected (0.01 mg/g DW). The average artemether in transgenic lines of A. dubia was
lower than A. annua this was 2 mg/g DW in shoots compared to control shoots (0.3
mg/g DW), transformed plant roots (0.7 mg/g DW) and hairy roots (1.62 mg/g DW),
whereas negligible amount of artemether was found in control roots of A. dubia (Fig.
3.12). In general, higher amount of artemether was observed in transgenic plants of
Artemisia annua as compared to transgenic plants of Artemisia dubia (Fig. 3.16).
The average arteether content from three transgenic lines in the shoots of
Artemisia annua was 7 mg/g dry weight (DW) compared to 1.5mg/g DW in the
controls shoots. Roots of transformed plants had an average arteether content of 1.6
mg/g DW while the amount found in transformed hairy roots was significantly greater
(3.6 mg/g DW) compared to the control roots in which negligible amounts could be
detected (0.016 mg/g DW). The average arteether in transgenic lines of A. dubia was
lower than A. annua this was 3.16 mg/g DW in shoots compared to control shoots
(0.1mg/g DW), transformed plant roots (0.75 mg/g DW) and hairy roots (1.61 mg/g
DW), whereas negligible amount of arteether was found in control roots of A. dubia
(Fig. 3.13). In general, higher amount of arteether was observed in transgenic plants
of Artemisia annua as compared to transgenic plants of Artemisia dubia (Fig. 3.16).
The average Dihydro-artemisinin content from three transgenic lines in the
shoots of Artemisia annua was 8.9 mg/g dry weight (DW) compared to 2.2 mg/g DW
in the controls shoots. Roots of transformed plants had an average dihydro-artemisinin
content of 2.5 mg/g DW while the amount found in transformed hairy roots was
significantly greater (6.8 mg/g DW) compared to the control roots in which negligible
amounts could be detected (0.03 mg/g DW). The average artemisitene in transgenic
lines of A. dubia was lower than A. annua this was 3.5 mg/g DW in shoots compared
to control shoots (0.25 mg/g DW), transformed plant roots (0.91 mg/g DW) and hairy
roots (1.53 mg/g DW), whereas negligible amount of dihydro-artemisinin was found
in control roots of A. dubia (Fig. 3.14). In general, higher amount of dihydroartemisinin was observed in transgenic plants of Artemisia annua as compared to
transgenic plants of Artemisia dubia (Fig. 3.16).
The average artesunate content from three transgenic lines in the shoots of
Artemisia annua was 5.5 mg/g dry weight (DW) compared to 1.04 mg/g DW in the
controls shoots. Roots of transformed plants had an average artesunate content of 0.86
mg/g DW while the amount found in transformed hairy roots was significantly greater
(2.6 mg/g DW) compared to the control roots in which negligible amounts could be
detected (0.006 mg/g DW). The average artesunate in transgenic lines of A. dubia was
lower than A. annua this was 2.16 mg/g DW in shoots compared to control shoots
(0.10 mg/g DW), transformed plant roots (0.61 mg/g DW) and hairy roots (1.44 mg/g
DW), whereas negligible amount of artesunate was found in control roots of A. dubia
(Fig. 3.15). In general, higher amount of artesunate was observed in transgenic plants
of Artemisia annua as compared to transgenic plants of Artemisia dubia (Fig. 3.16).
(a)
(b)
(C)
Fig. 3.4: HPLC Chromatograms of (a) Standard Artemether (b) Artemisia annua
Sample (A2) (c) Spike Sample (A.annua sample + standard Artemether)
(a)
(b)
©
Fig. 3.5: HPLC Chromatograms of (a) Standard Artemether (b) Artemisia dubia
Sample (D1) (c) Spike Sample (A.dubia sample + standard Artemether)
(a)
(b)
(C)
Fig. 3.6: HPLC Chromatograms of (a) Standard Arteether (b) Artemisia annua
Sample (A2) (c) Spike Sample (A.annua sample + standard Arteether)
(a)
(b)
(C)
Fig. 3.7: HPLC Chromatograms of (a) Standard Arteether (b) Artemisia dubia
Sample (D1) (c) Spike Sample (A.dubia sample + standard Arteether)
(a)
(b)
(C)
Fig. 3.8: HPLC Chromatograms of (a) Standard Dihydroartemisinin (b)
Artemisia annua Sample (A2) (c) Spike Sample (A.annua sample + standard
Dihydroartemisinin)
(a)
(b)
(C)
Fig. 3.9: HPLC Chromatograms of (a) Standard Dihydroartemisinin (b)
Artemisia dubia Sample (D1) (c) Spike Sample (A.dubia sample + standard
Dihydroartemisinin)
(a)
(b)
(C)
Fig. 3.10: HPLC Chromatograms of (a) Standard Artesunate (b) Artemisia
annua Sample (A2) (c) Spike Sample (A.annua sample + standard Artesunate)
(a)
(b)
(C)
Fig. 3.11: HPLC Chromatograms of (a) Standard Artesunate (b) Artemisia dubia
Sample (D1) (c) Spike Sample (A.dubia sample + standard Artesunate)
8
Artesunate Content (mg/g) DW
7
6
5
4
3
2
1
0
Fig. 3.12: Artemether content in shoots roots and hairy roots of A.annua.A1, A2
and A3 represents leaf samples from three different transgenic lines, AR1, AR2
and AR3 represents roots of transgenic lines A1, A2 and A3 respectively, AH1
and AH2 represents two transgenic lines of hairy roots, AC and ARC represent
control shoots and roots. A2+Artemether and D2+ Artemether are the spike
samples used for verification of artemether concentration.
10
Arteether content (mg/g) DW
9
8
7
6
5
4
3
2
1
0
Fig. 3.13: Arteether content in shoots roots and hairy roots of A.annua. A1, A2
and A3 represents leaf samples from three different transgenic lines, AR1, AR2
and AR3 represents roots of transgenic lines A1, A2 and A3 respectively, AH1
and AH2 represents two transgenic lines of hairy roots, AC and ARC represent
control shoots and roots. A2+Arteether and D2+ Arteether are the spike samples
used for verification of arteether concentration.
12
Dihydro-artemisinin content (mg/g) DW
10
8
6
4
2
0
Fig. 3.14: Dihydro-artemisinin content in shoots roots and hairy roots of
A.annua.A1, A2 and A3 represents leaf samples from three different transgenic
lines, AR1, AR2 and AR3 represents roots of transgenic lines A1, A2 and A3
respectively, AH1 and AH2 represents two transgenic lines of hairy roots, AC
and ARC represent control shoots and roots. A2+Dihydroartemisinin and D2+
Dihydroartemisinin are the spike samples used for verification of
dihydroartemisinin concentration.
8
Artesunate Content (mg/g) DW
7
6
5
4
3
2
1
0
Fig. 3.15: Artesunate content in shoots roots and hairy roots of A.annua.A1, A2
and A3 represents leaf samples from three different transgenic lines, AR1, AR2
and AR3 represents roots of transgenic lines A1, A2 and A3 respectively, AH1
and AH2 represents two transgenic lines of hairy roots, AC and ARC represent
control shoots and roots. A2+Arteether and D2+ Arteether are the spike samples
used for verification of arteether concentration.
Contents of Artemisinin and its Derivatives (mg/g) DW
50
A
45
Artemisinin
Artemether
Dihydro-artemisinin
Artesunate
Arteether
40
35
30
25
20
B
15
10
5
C
CD
EF EF FGHI
FGHIJGHIJKL
FGHIJKL
KLHIJKL EFGH
EFGH M
HIJKL
D
EFGH
EFGFGHI
FGHIJ
FGHI
GHIJKLFGHIJK
FGHIJK
N N
IJKLKLKLJKLL
0
Fig. 3.16: Comparative and Statistical analysis of Average Artemisinin,
Artemether, Arteether, Dihydro-artemisinin and Artesunate content in shoots,
roots and hairy roots of transformed and un-transformed plants of A.dubia and
A.annua. Each value is the mean of three replicates. Any two means having a
common alphabet are not significantly different at p = 0.05 using LSD. Vertical
bar represents the standard error of the 3 means.
IJKL
HIJKL
IJKL
IJKL
JKLM
3.3.3: Statistical Analysis
3.3.3.1: Comparative analysis of artemisinin and its derivatives in different
tissues of Artemisia annua and Artemisia dubia
Statistical analysis was conducted in factorial design (2 X 5 X 3) to see the
effect of different tissues on enhanced production of artemisinin and its derivatives in
different tissues of A.annua and A.dubia.
Artemisia annua and Artemisia dubia had a significant difference (P<0.05) in
production of artemisinin and its derivatives. Most effective results were obtained in
case of different tissues of A.annua species (Table 3.1).
Table 3.1: Analysis of Variance Table for factors affecting Production of
Artemisinin and its Derivatives in different tissues of A.annua and A.dubia
Source of Variation
df
Sum of Squares
Mean Square
F-Value
Prob.
Species
1
743.849
743.849
445.5682
0.0000
4
1225.680
306.420
183.5467
0.0000
4
1098.772
274.693
164.5421
0.0000
Tissues
2
881.418
440.709
263.9862
0.0000
Species X Tissues
2
407.269
203.635
121.9780
0.0000
8
1012.384
126.548
75.8028
0.0000
8
970.030
121.254
72.6314
0.0000
Error
60
100.166
1.669
Total
89
6439.567
Artemisinin and its
Derivatives
Species X
Artemisinin and its
Derivatives
Tissue X Artemisinin
and its Derivatives
Species X Tissues X
Artemisinin and its
Derivatives
Coefficient of Variation: 27.35%
Rol genes had significant impact (P<0.05) on production of artemisinin and its
derivatives. Mean value of artemisinin was higher in all plants tissues as compared to
other derivatives of artemisinin. Generally artemisinin content was more enhanced in
all tissues of both Artemisia species. Similarly, different tissues had significant impact
(P<0.05) on production of artemisinin and its derivatives in A.annua and A.dubia.
Mean value of artemisinin and its derivatives was higher in shoots of both species as
compared to other tissues (Table 3.1).
In general Artemisia annua is the best species for production of artemisinin and
its derivatives, and among all metabolites artemisinin was most enhanced metabolite
compared to other metabolites under the effect of transformation with rol genes.
Shoots are the best tissues where production of these metabolites was more enhanced
compared to other tissues. Among roots, hairy roots of both species produced more
artemisinin and its derivatives compared to other roots.
3.4: Conclusion
In this part, Comparative analysis of artemisinin content and its derivatives in
transformed and untransformed plants of Artemisia annua and Artemisia dubia was
carried out and following conclusion were drawn:
Artemisinin was significantly increased in the transformed shoots, hairy
roots and roots of transformed shoots compared to control in both A.annua
and A.dubia plants.
Highest amount of artemisinin was found in transgenic shoots of
transformed A.annua and A.dubia (increased by a factor of 10) compared
to control shoots of both species. Roots of these transformed shoots also
contain significantly increased amount of artemisinin compared to control
roots.
Significant amount of artemisinin was observed in hairy roots of both
A.annua and A.dubia compared to control roots.
Artemether, arteether, dihydroartemisinin and artesunate contents were
also significantly increased in transgenic shoots of both Artemisia species
compared to control shoots. Amount found in roots of transformed shoots
and transformed hairy roots was significantly greater compared to the
control roots in which negligible amounts was detected.
In general, results showed that artemisinin content was enhanced much
more as compared to its derivatives under the effect of transformation with
rol genes and among two Artemisia species, A.annua is the best species for
the production of artemisinin and its derivatives. Considering the tissue
types, shoots and hairy roots are more appropriate for production of
artemisinin and its derivatives.
Chapter No. 4
Analysis of Metabolic pathway and Trichome development and their comparison
in transformed and un-transformed plants of Artemisia annua
and Artemisia dubia
4.1: Introduction
Artemisinin is a secondary metabolite that has been found to have strong
antimalarial activity with little or no side effects (Klayman, 1985). Artemisinin is a
sesquiterpene lactone which has been reported to be produced within the glandular
trichomes of Artemisia species, a member of the Asteraceae family. The current
understanding is that artemisinin is produced in 10-celled glandular trichomes located
on leaves, floral buds, and flowers (Ferreira et al., 1995; Tellez et al., 1999; Olsson et
al., 2009) and sequestered in the epicuticular sac at the apex of the trichome (Olsson
et al., 2009). The evidence in its support is the observation that, artemisinin
concentrations are higher in leaves that are formed later in development than in leaves
formed early in the plant‘s development; this difference has been attributed to a
higher trichome density and a higher capacity per trichome in the upper leaves
(Lommen et al., 2006). It has been shown that hairy roots produced by infection with
A.rhizogenes can produce artemisinin (Jaziri et al., 1995; Liu et al., 1999). This would
suggest that the plant may be capable of producing artemisinin in the absence of
trichomes.
There are two independent and differentially-localized pathways involved in
terpene biosynthesis that converge to yield a common pool of the terpenoids precursor
called isopentenyl diphosphate (IPP) (Croteau et al., 2000; Lange et al., 2000) (Figure
1.15). The mevalonic acid pathway (MVA) which initiates from acetyl-CoA is located
in the cytosol. The key, regulatory, step in this pathway is the conversion of
hydroxymethylglutaryl-CoA (HMG) to mevalonate via, a regulatory enzyme,
hydroxymethylglutaryl-CoA reductase (HMGR). Several subsequent steps lead to
formation of the cytosolic localized pool of IPP (Towler and Weathers, 2007). The
other pathway, non-mevalonate pathway (MEP), to IPP begins with pyruvate and
occurs in the plastid with no mevalonate intermediate. Several subsequent steps
synthesize the plastid pool of IPP (Rohmer et al., 1996).
Numerous investigations have shown that the Agrobacterium rol genes can
induce high levels of secondary metabolites in hairy root cultures of most transformed
plant species (Giri and Narasu, 2000; Sevon and Oksman-Caldentey, 2002). We
hypothesized that transformation with the rol genes could induce an increase in
artemisinin through stimulation of the synthesis pathways. Hence, the aim of the
investigation reported here was to compare trichome densities in transformed and
untransformed plants of Artemisia annua and Artemisia dubia and the genes involved
in the synthesis pathway were monitored to ascertain how the rol genes could act to
influence the production of artemisinin.
4.2: Materials and Methods
4.2.1: Analysis of metabolic pathway
Young leaves, roots and hairy roots were collected separately from 3-4 months
old plants, frozen in liquid nitrogen, ground to a fine powder with electrical
homogenizer and used for RNA extraction.
4.2.1.1: RNA extraction
RNA extraction was performed using RNAqueous Small Scale Phenol-Free
Total RNA Isolation Kit (Ambion) for small scale RNA isolation according to the
manufacturer‘s instructions. Frozen plant tissue powder (70 mg) was used for each
RNA extraction.
Before working with RNA, lab bench, and pipettes were cleaned with an
RNAase decontamination solution such as Ambion RNAase Zap RNAase
decontamination solution
70 mg of frozen leaves, roots and hairy roots were weighed and stored at 20ºC for two or three days before extraction.
Elution solution (typically 50-200µl per prep) was pre-heated in an RNAase
free microcentrifuge tube in the water bath at 70-80 ºC before starting the
extraction.
Samples were ground with freeze electrical grinder, and Lysis / Binding
solution in the ratio of 50mg: 600µl was added after grinding.
After adding the solution, samples were centrifuged for 10.minutes at
13,000rpm to remove debris.
After centrifugation, supernatant was transferred to new tube and an equal
volume of 64% Ethanol was added and mixed by vortexing.
(a) Lysate /Ethanol mixture was drawn through a filter cartridge.
(b) The Maximum volume that can be applied at one time is 700µl; it was
centrifuged for 30.second-1 minute at 13000rpm until the lysate/ethanol
mixture was through the filter
(c) Flow through was discarded and collection tube was reused for the
washing steps
700µl Wash solution # 1 was added to the filter cartridge and centrifuged for
30.second, flow through was discarded and tube was reused for subsequent
washes.
500µl Wash solution # 2/3 was added and centrifuged for 30.second. Filter
cartridge was placed into new collection tube.
Pre-heated elution solution was added to the filter cartridge in two steps, (i)
first by adding 40 µl and then centrifuged for 1.minute (ii) second with 30 µl
and again centrifuged for 1 minute.
Quality and quantity of RNA was checked at 260 nm by Nanodrop and
samples were stored at -20ºC in refrigerator.
Quality of RNA was analyzed by performing RNA 6000 Nano Assay by using
Agilent RNA 6000 Nano Assay Kit and Bioanalyzer (Agilent Technologies,
Germany,
reorder
number
5067-1511)
according
to
manufacturer‘s
instructions.
4.2.1.2: cDNA synthesis
RNA (1 µg) was reverse transcribed using ThermoScript RT-PCR system
(Invitrogen) primed with oligo (dT) 20 primer. The RNA was removed from the first
strand cDNA by RNase treatment using RNase H (Invitrogen) according to the
manufacturer‘s instructions.
cDNA was prepared from RNA by the following method:
Table 4.1: Different components used for Sample preparation
Amount used for
Amount used for
1 reaction
12 reactions
S.No.
Components
1
DEPC Water
8µl
96µl
2
5mMPrimer (Oligo dT)
1µl
12µl
3
10mM RNA sample
1µl
12µl
4
10mM dNTPs mix
2µl
24µl
All the mixture (prepared above) was denatured by incubating at 65ºC for 5
minutes in pre-heated water bath at 65 ºC and placed on ice after 5 minutes.
Then reaction mixture was prepared for cDNA Synthesis, 5X cDNA Buffer was
vortexed for 5 second just prior to use.
Master Mix was prepared and all reactions were performed on ice during this
synthesis.
Then the reaction mixture was made as:
Table 4.2: Different components used for Master Mix for cDNA Synthesis
Amount used for 1
Amount used for 12
reaction
reactions
5x cDNA Synthesis Buffer
4µl
48µl
2
0.1 M DTT
1µl
12µl
3
RNAase OUT
1µl
12µl
4
DEPC Water
1µl
12µl
5
Thermo script RT
1µl
12 µl
S.No.
Components
1
8 µl of reaction mixture was added into the each tube containing sample.
Samples were transferred to the preheated thermo cycler to the appropriate
cDNA Synthesis temperature and incubated as follows: (1) 60 minutes at 50ºC
(2) 85ºC for 5 minutes.
1 µl of RNAase H was added to the samples and incubated at 37 ºC for
20.minutes.
Samples were stored at -20 ºC immediately after preparation of cDNA or used
for PCR immediately.
4.2.2: Quantitative real time polymerase chain reaction (qRT-PCR)
The real-time RT-PCR analysis was performed in a LightCycler® 480 RealTime PCR System, version 1.5 (Roche Applied Sciences, Mannheim, Germany),
using the cDNAs as templates and the random primers for the analyzed gene, as listed
in Table 4.3. The actin gene was used as a reference gene. SYBR Green LightCycler®
480 DNA SYBR Green I Master (Roche Applied Sciences, Mannheim, Germany,
Cat. No. 04707516001) was used in the Polymerase Chain Reaction (PCR) to quantify
the amount of dsDNA. PCR was performed using 2.5 – 3µl of cDNA in a total of 20µl
reaction volume. The PCR conditions were 2 minutes at 95ºC, 30 seconds at 95ºC, 30
seconds at 60-62ºC, 1 minute at 72ºC for 40 cycles, followed by 5 minutes at 72ºC.
The conditions were selected because none of the samples analyzed reached a plateau
at the end of the amplification (i.e. they were at the exponential phase of the
amplification).
Table 4.3: Primers used for qRT-PCR
No
Name
1
TFAR1
2
ADS
3
ALDH1
4
CYP71AV1
5
ACTIN
Forward Primer
Reverse Primer
CCTTGGAGATCCTGAA
CGTTGGATTGTGCTGAAC
GCTG
TG
GGGAGATCAGTTTCTC
CTTTTAGTAGTTGCCGCA
ATCTATGAA
CTTCTT
CAGGAGCTAATGGAA
TTTCTTCCTTCGGCCACTG
GTTCTAAGTCAG
TTG
AGGGTAGGCATTCGCC
TCGAGTGGCCCTAACAAC
GTCC
CTGC
ATCAGCAATACCAGG
AGGTGCCCTGAGGTCTTG
GAACATAGT
TTCC
Accession
Numbers
GU733320
JQ319661
FJ809784
HQ315834
EU531837
Expression analysis of each gene was confirmed in at least 3 independent RTreactions using forward and reverse primers and expression levels of each gene was
normalized using actin gene as a control.
Expression of genes was also analyzed by using agarose gel electrophoresis. 1.5
percent w/v agarose gel was prepared by melting 1.5 gm of agarose in 100 ml of 1 X
TBE buffer in microwave oven. 4 µl (4 µl/ 100 ml) gel red solution was added to stain
DNA. DNA samples were mixed with DNA loading dye containing 0.25%
bromophenol blue prepared in 40% sucrose solution. Electrophoresis was performed
at 100 volts (50 mA) for 65 minutes in 1 X TBE running buffer. After electrophoresis
amplified product was detected by placing the gel on UV-Trans illuminator (Life
Technology, USA).
4.2.3: Analysis of trichome density
The number of glandular trichomes was determined at the adaxial side of 3
random pieces of fresh leaf material from each sample with an accurately determined
leaf area of approximately 5mm2per leaf. Glandular trichomes were counted by using
environmental scanning electron microscope (ESEM) at 200x magnification. Leaves,
roots and hairy roots were cut from the transformed and non-transformed plants of
Artemisia annua and Artemisia dubia using a scalpel and transferred to the
microscope chamber for trichome analysis.
4.2.3.1: Environmental scanning electron microscope (ESEM) setup
The sample was placed, upper epidermis upwards, on adhesive carbon tape fixed
to a 10 mm stainless steel mounting disc in thermal contact with water cooled Peltier
chip in the sample chamber. This allowed temperature control of the whole sample
and meant that complete leaves or leaf tips could be accommodated, minimizing the
number of potentially dehydrating cut edges. All experiments were conducted using
an FEI XL30 Tungsten filament ESEM with the Secondary electron signal collected
using a gaseous secondary electron detector. The accelerating voltage was 10-15kV.
A water vapour pressure of 5mBar was used to minimize sample evaporation and
samples were maintained at 2 °C. The time elapsing between cutting the leaf tissue
and capturing the first image was minimized such that the tissue was as fresh as
possible; this preparation and pump down time typically ranged from two to five
minutes.
4.3: Results
4.3.1: Analysis of metabolic pathway and trichome development
The genes ADS, CYP71AV1 and ALDH1 are involved in the metabolic
synthesis pathway of artemisinin and are involved in the conversion of FDP to
dihydroartemisinic acid, which is a late precursor of artemisinin (Fig. 1.15). These
key genes were used to determine how transformation with the rol genes affected the
pathway in the different tissues of A. annua and A. dubia. Trichome-Specific fatty
acyl-CoA reductase 1(TAFR1) has been suggested to be involved in trichome
development and sesquiterpenoid biosynthesis, both of which are important for
artemisinin production. The expression of this gene was monitored to determine if
trichome density might also be affected.
4.3.2: Relative expression of genes involved in metabolic pathway of artemisinin
biosynthesis
Pronounced changes in the expression of the artemisinin biosynthetic pathway
genes were observed in all transgenic lines of A. annua and A. dubia transformed with
rol genes (Fig. 4.1 A,B,C,D,E). The qPCR data presented here clearly demonstrate
that the expression levels of ADS, CYP71AV1 and ALDH1 were significantly
increased (P < 0.0001) in transformed plants of both Artemisia species compared to
untransformed plants. Expression of artemisinin biosynthetic pathway genes was
observed in hairy roots and roots of transformed plants compared to control roots of
both A. annua and A. dubia in which low or negligible levels of expression were
found. Expression levels of all the artemisinin biosynthetic genes were also
significantly higher in transformed shoots compared to control plants. Interestingly
transformed A. dubia showed levels, which were greater than, control A. annua plants
despite similar artemisinin content. Not all the genes examined exhibited the same
increase in expression. Cyp71AV1 appeared to show the greatest changes in relative
expression in both shoots and hairy roots of both A. annua and A. dubia compared to
the other genes. Its expression was significantly increased in transformed plants of
both Artemisia species compared to untransformed plants, i.e. ~ 5 and ~ 120 times
more in transformed leaves of A. annua and A. dubia, respectively. Roots of
transformed plants in both Artemisia species showed significantly higher expression
of this gene compared to control roots. The greatest difference in expression of this
gene was found in hairy roots from both species even compared to transformed roots
and control roots.
Relative expression of the ADS gene was generally lower than that of the other
genes however the difference in its expression compared to the control was the
highest. i.e. ~ 11 and ~ 270 times greater in transformed leaves of A. annua and A.
dubia respectively. Roots from these plants showed even larger differences in the
expression of this gene compared to the controls. In A.annua the levels of expression
of ADS was comparable to transformed roots and hairy roots but in A.dubia there was
a significant difference in the expression of this gene.
The ALDH1 gene showed high expression in transformed plants of both
Artemisia species when compared to untransformed plants, i.e. ~ 7 and ~ 5 times more
in transformed leaves of A. annua and A. dubia, respectively. Roots of these
transformed plants also showed greater differences in expression of this gene in A.
annua compared to control, in contrast in A.dubia the level of expression of this gene
was comparatively low. However expression of this gene showed significant
differences in hairy roots of both Artemisia species compared to control roots.
4.3.3: Trichome development
A significant increase in expression of TFAR1 was detected in transformed
plants of both Artemisia species compared to untransformed plants, i.e. ~ 10 and ~
300 times more in transformed leaves of A. annua and A. dubia, respectively.
Unexpectedly this gene was expressed in abundance in transformed roots and hairy
roots of both Artemisia species. The levels of expression were greater than that seen
in control shoots. Although this gene has been suggested to be responsible for
trichome density it showed significant differences in expression even in transformed
roots and hairy roots compared to control roots figure 4.1 (A).
Relative expression of TFAR1 gene
10
9
8
7
6
5
4
3
2
1
0
Fig. 4.1 (A): Comparative analysis of expression of TFAR1 gene in shoots, roots
of transformed plants and hairy roots of transformed and un-transformed plants
of Artemisia dubia and Artemisia annua. The average of three plants is shown
together with error bars showing SE.
Relative expression of ADS gene
7
6
5
4
3
2
1
0
Fig. 4.1 (B): Comparative analysis of expression of ADS gene in shoots, roots of
transformed plants and hairy roots of transformed and un-transformed plants of
Artemisia dubia and Artemisia annua. The average of three plants is shown
together with error bars showing SE.
Relative expression of CYP71AV1 gene
12
10
8
6
4
2
0
Fig. 4.1 (C): Comparative analysis of expression of CYP71AV1 gene in shoots,
roots of transformed plants and hairy roots of transformed and un-transformed
plants of Artemisia dubia and Artemisia annua. The average of three plants is
shown together with error bars showing SE.
10
Relative expression of ALDH1 gene
9
8
7
6
5
4
3
2
1
0
Fig. 4.1 (D): Comparative analysis of expression of ALDH1 gene in shoots, roots
of transformed plants and hairy roots of transformed and un-transformed plants
of Artemisia dubia and Artemisia annua. The average of three plants is shown
together with error bars showing SE.
4.3.4: Statistical analysis
4.3.4.1: Comparative analysis of different genes involved in artemisinin
production pathway in different tissues of Artemisia annua and Artemisia dubia
Statistical analysis was conducted in factorial design (2 X 3 X 4) to see the
effect of rol ABC genes on metabolic pathway artemisinin production and different
genes involved in this pathway in different tissues of A.annua and A.dubia.
Artemisia annua and Artemisia dubia had a significant difference (P<0.05) in
production of artemisinin. Most effective results were obtained in case of different
tissues of A.annua species. All genes were expressed more in A.annua plant.
Table 4.4: Analysis of Variance Table for factors affecting different genes
involved in artemisinin production pathway in different tissues of A.annua and
A.dubia
Source of Variation
df
Sum of Squares
Species
1
61.624
Tissue Types
2
Mean
F-Value
Prob.
61.624
75.9609
0.0000
197.943
98.971
121.9981
0.0000
2
40.760
20.380
25.1215
0.0000
Genes
3
33.288
11.096
13.6775
0.0000
Species X Genes
3
4.236
1.412
1.7404
0.1713
6
30.181
5.030
6.2006
0.0001
6
5.571
0.928
1.1445
0.3516
Error
48
38.940
38.940
0.811
Total
71
412.542
Species X Tissue
Types
Tissue Types X
Genes
Species X Tissue
Types X Genes
Square
Coefficient of Variation: 35.81%
Effect of different tissues had significant impact (P<0.05) on production of
artemisinin in A.annua and A.dubia. Mean value of artemisinin was higher in shoots
of both species as compared to other tissues. Similarly, different genes of artemisinin
synthesis pathway had significant impact (P<0.05) on production of artemisinin. In
general CYP71AV1 gene was highly expressed as compared to other genes of
pathway of artemisinin synthesis (Table 4.4).
In general Artemisia annua is the best species for production of artemisinin,
and among all genes expression of CYP71AV1 gene was higher in both species as
compared to other genes under the effect of transformation with rol genes. Shoots are
the best tissues where production of artemisinin was more enhanced compared to
other tissues. Among roots, hairy roots of both species produced more artemisinin and
its derivatives compared to other roots (Table 4.4).
8
B
A
9
CYP
ALDH1
C
ADS
D
6
HI
EFGHI
HI
M
M
M
BCD
CDEF
BCD
L
FGHI
BCD
CDEFG
DEFG
EFGHI
M
BCD
CDEF
J
DEFG
CDEF
JK
1
EFGHI
DEFG
HI
EFGHI
2
HI
3
CDE
CDEF
4
CDE
BC
CDE
BCDE
5
EFGHI
Relative Expression of Genes
7
TFAR1
0
A.annua
Transformed
Shoots
A.annua
Control
Shoots
A.annua
Transformed
Roots
A.annua
Control
Roots
A.annua
A.dubia
Hairy Roots Transformed
Shoots
A.dubia
Control
Shoots
A.dubia
Transformed
Roots
A.dubia
Control
Roots
Fig 4.2: Statistical and comparative analysis different genes involved in
artemisinin production pathway in different tissues of Artemisia annua and
Artemisia dubia. Each value is the mean of three replicates. Any two means
having a common alphabet are not significantly different at p = 0.05 using LSD.
Vertical bar represents the standard error of the 3 means.
A.dubia
Hairy Roots
4.3.4: Analysis of trichome density
The number of glandular trichomes was determined at the adaxial side of 3
random pieces of fresh leaf material from each sample with an accurately determined
leaf area of approximately 5mm2 per leaf. Glandular trichomes were counted by using
environmental scanning electron microscope (ESEM) at 200x magnification. Leaves,
roots and hairy roots were cut from the transformed and non-transformed plants of
Artemisia annua and Artemisia dubia using a scalpel and transferred to the
microscope chamber for trichome analysis.
Trichome density was assessed using ESEM at low resolution and is shown in
Figure 4.3 A and B. Transformed leaves of A.annua plants produced more trichomes
(~ 222 trichomes/5mm2) as compared to the control leaves (~ 120 trichomes/5mm2)
(Fig. 4.3A). Transformed leaves of Artemisia dubia also produced more trichomes (~
173 trichomes/5mm2) compared to control leaves (~ 90 trichomes/5mm2). In contrast,
the hairy roots, transformed and untransformed roots from both Artemisia species
showed no trichome production (Fig. 4.3 A).
Transformation also increased hairiness in transformed plants along with
increase in trichome density compared to non transformed plants. Transformed plants
produced denser network of hair on their surface compared to non-transformed plants
(Fig. 4.3 B).
Table.4.5: Analysis of Variance for significant difference between different
species of Artemisia plant
Degrees of
Freedom
Sum of Squares
Mean
Square
Between
7
18652.292
2664.613
Within
16
140.667
8.792
Total
23
18792.958
Coefficient of Variation = 1.62%
F-Value
Prob.
303.084
0.0000
250
Number of adaxial Trichomes
per 5mm2 leaf
A
200
150
100
B
C
C
D
D
E
F
50
0
Fig. 4.3 (A): Comparative analysis of trichome density in shoots roots and hairy
roots of A.annua and A.dubia. A1, A2 and A3 represents three different
transgenic lines of shoots, AR1, AR2 and AR3 represents transformed roots of
transgenic lines A1, A2, A3 respectively, AH1 and AH2 represents two
transgenic lines of hairy roots, AC and ARC represent control shoots and roots
of respectively. D1, D2 and D3 represents three transgenic lines of leaves, DR1,
DR2 and DR3 represents transformed roots of transgenic lines D1, D2, D3
respectively, DH1 and DH2 represents two transgenic lines of hairy roots, DC
and DRC represent control shoots and roots of respectively. The average of three
plants is shown together with error bars showing SE. Data was statistically
analyzed for significant difference by using ANOVA and Duncan’s Multiple
Range Test. Each value is the mean of three replicates. Any two means having a
different alphabet are significantly different at p = 0.05 using LSD.
(A1)
(A2)
(A3)
(A). Transformed shoots of Artemisia annua
(D1)
(D2)
(D3)
(B). Transformed Leaves of Artemisia dubia
(AC)
(DC)
(C). Un-transformed Leaves of Artemisia annua and Artemisia dubia
(
(AR1)
(AR2)
(AR3)
(A). Transformed Roots of Artemisia annua
(DR1)
(DR2)
(DR3)
(B). Transformed Roots of Artemisia dubia
(ARC)
(DRC)
(C). Un-transformed Roots of Artemisia annua and Artemisia dubia
(AH1)
(AH2)
(A). Hairy Roots of Artemisia annua
(DH1)
(DH2)
(B). Hairy Roots of Artemisia dubia
Figure: 4.3 (B). Comparison of trichome density in different tissues of
transformed and untransformed plants of A.annua and A.dubia. A1, A2 and A3
represents three transgenic lines of leaves in A.annua, AR1, AR2 and AR3
represents transformed roots of transgenic lines A1, A2 and A3 respectively,
AH1 and AH2 represents two transgenic lines of hairy roots, AC and ARC
represent control shoots and roots of A.annua respectively. D1, D2 and D3
represents three transgenic lines of leaves in A.dubia, DR1, DR2 and DR3
represents transformed roots of transgenic lines D1, D2 and D3 respectively,
DH1 and DH2 represents two transgenic lines of hairy roots, DC and DRC
represent control shoots and roots of A.dubia respectively. Both technical and
biological replicates were performed.
4.4: Conclusion
In this part, metabolic pathway of artemisinin production in Artemisia
annua and Artemisia dubia and the effect of rol genes through which these genes
enhance the production of artemisinin were analyzed using different genes involved in
artemisinin production and TFAR1 involved in trichome development and
sesquiterpenoid biosynthesis was also studied. Trichome density was also calculated
in both Artemisia species and following conclusion were drawn:
Pronounced changes in the expression of the artemisinin biosynthetic
pathway genes were observed in all transgenic lines of A. annua and A.
dubia transformed with rol ABC genes. Expression levels of ADS,
CYP71AV1 and ALDH1 were significantly increased in transformed
plants and hairy roots of both Artemisia species compared to
untransformed plants.
Cyp71AV1 appeared to show the greatest changes in relative expression in
both shoots and hairy roots of both A. annua and A. dubia compared to the
other genes. Roots of transformed plants in both Artemisia species showed
significantly higher expression of this gene compared to control roots. The
greatest difference in expression of this gene was found in hairy roots from
both species even compared to transformed roots and control roots.
Relative expression of the ADS gene was generally lower than that of the
other genes however the difference in its expression compared to the
control was the highest. Roots from these plants and hairy roots showed
even larger differences in the expression of this gene compared to the
controls.
The ALDH1 gene showed high expression in transformed plants of both
Artemisia species when compared to untransformed plants. Roots of these
transformed plants also showed greater differences in expression of this
gene in A. annua compared to control, in contrast in A.dubia the level of
expression of this gene was comparatively low. However expression of
this gene showed significant differences in hairy roots of both Artemisia
species compared to control roots.
A significant increase in expression of TFAR1 was detected in
transformed plants of both Artemisia species compared to untransformed
plants. Unexpectedly this gene was expressed in abundance in transformed
roots and hairy roots of both Artemisia species.
Transformed leaves of A.annua and A.dubia plants produced more
trichomes as compared to the control leaves. In contrast, the hairy roots,
transformed and untransformed roots from both Artemisia species showed
no trichome production.
In general, results showed that among different genes involved in
artemisinin synthesis and trichome development CYP71AV1 was highly
expressed compared to other genes of metabolic pathway and among two
Artemisia species, A.annua is the best species for the production of
artemisinin. Considering the tissue types, shoots and hairy roots are more
appropriate for production of artemisinin.
Chapter No. 5
Analysis of anticancer activity on Breast Cancer Cell lines and comparison in transformed
and untransformed Artemisia annua and Artemisia dubia
5.1: Introduction
Cancer is a foremost cause of death all over the world. According to different
data published, it is described that cancer caused 7.9 million deaths (around 13 % of
all deaths) in 2007 and this number is increasing every day with an estimation of 12
million deaths in 2030. The different types of cancer which cause maximum number
of deaths every year include lung, stomach, liver, colon and breast cancer. Cancer
cells are produced due to changes in cell acquired by external agents or by inherited
genetic factors. According to World Health Organization deaths due to cancer can be
prevented up to 30% (WHO, 2008d).
A large number of studies carried out by different laboratories all over the
world now explain the effect of artemisinin and its derivatives on different types of
cancer, such as breast cancer (Posner et al., 2004; Nam et al., 2007; Sing and Lai,
2004; Thomas et al., 2003), lung cancer (Wu et al., 2006; Thomas et al., 2003),
prostate cancer (Chen et al., 2004; Thomas et al., 2003),), head and neck cancer
(Yamachika et al., 2004), bladder carcinomas, renal carcinoma (Thomas et al., 2003),
ovarian carcinoma (Jiao et al., 2007), cervical carcinoma (Disbrow et al., 2005),
pancreas carcinoma (Wu et al., 2001), colon carcinoma (Thomas et al., 2003), thyroid
medullary carcinoma, endometrial carcinoma and oral squamous cell carcinoma
(Yamachika et al., 2004).
To date, considerable pharmacological studies on anticancer use of artemisinin
have been carried out which explain mechanism of action of artemisinin against
cancer in very effective way (Efferth et al., 2001; Singh and Lai, 2001; Efferth et al.,
2002; Sadava et al., 2002). According to these considerations, cancer cells require
large amount of iron for their multiplication and transferrin receptors that facilitate
uptake of the plasma iron-carrying protein transferrin via endocytosis, and a large
concentration of cell surface to express, artemisinin reacts with iron to form free
radicals which can kill cells. By covalently tagging artemisinin to transferrin,
artemisinin could be selectively picked up and concentrated by cancer cells. In
addition, both artemisinin and iron would be transported into the cell in one package.
Once an artemisinin-tagged transferrin molecule is endocytosed, iron is released and
reacts with tagged artemisinin. As a result, free radicals form that kill the cancer cells.
Artemisinin-tagged transferrin is highly selective and potent in killing cancer cells.
Thus, artemisinin and artemisinin-tagged iron-carrying compounds could be
developed into potent anticancer drugs (Rowen, 2002; Henry et al., 2005).
The aim of the investigation reported here was to analyse the anticancerous
properties of transformed and non-transformed plants of Artemisia annua and
Artemisia dubia.
5.2: Materials and Methods
5.2.1: Analysis of transformed and untransformed A.annua and A.dubia on
breast cancer cell lines
Transformed and Un-transformed plants of Artemisia annua and Artemisia
dubia and hairy roots from these plants were analyzed to check their affectivity
against cancer by using MCF7 breast cancer cell lines. Plants were analyzed for
anticancer activity by using method of Vanicha and Kanyawim (2006)
Sulforhodamine B (SRB) antiproliferative assay. Standard artemisinin was used as
positive control.
5.2.2: Sample preparation
Artemisinin is highly soluble in hexane/ethyl acetate while it is least soluble in
water. To ensure the anticancerous activity of artemisinin, all samples were prepared
in two solvents that is hexane and aqueous, because A.annua and A.dubia plants also
contain secondary metabolites other than artemisinin.
5.2.2.1: Hexane extraction
Biomass extraction was performed according to the method by Lapkin et al.
(2006). Basically, 1g of transformed and untransformed A.annua and A.dubia plants
and hairy roots were extracted with 30ml hexane/ethyl acetate (9.5:0.5) at room
temperature in a Sonication bath (Kerry Ultrasonic UK) for 1 hour. Hexane was
modified with ethyl acetate to increase the solubility of artemisinin in the extraction
solvent. The extract was filtered over using Whatman 1.0 µm filter paper and
concentrated to dryness using a rota-evaporator (Buchi R-210, USA) at 40 ◦C. Then
these dried samples were dissolved in 100% (V/V) DMSO solution with 10 mg/ml for
analysis on breast cancer cell lines.
5.2.2.2: Aqueous extraction
100ml of boiling water was added to 1g of transformed and untransformed
A.annua and A.dubia plants and hairy roots, which was stirred and allowed to cool in
the dark for 30 min, based on a modification of Van der Kooy and Verpoorte (2011)
method. The filtered extract (Whatman 1 µm) was dried using a rota-evaporator
(Buchi R-210, USA) at 40 ◦C. 10mg of each dried sample was dissolved in 1ml of
100% (V/V) DMSO for analysis on breast cancer lines.
5.2.3: Cell preparation
5.2.3.1: Cells
The cell lines used in this study was MCF-7 human breast cancer cells. Details
of its origin and characterization are described elsewhere (Sanford et al., 1948; Gey et
al., 1952; Soule et al., 1973; Fogh and Trempelln, 1975). Cells grown as monolayer
cultures in T-75 flasks Costar were subcultured twice a week at 37◦C in an
atmosphere containing 5% CO2 in air and 100% relative humidity and maintained at
low pas- sage number (5 to 20). Cells were cultured in Dulbecco‘s modified Eagle‘s
medium DMEM, Warwick University, UK. Medium was supplemented with 10%
fetal bovine serum (FBS), 100 µg/ml streptomycin and 100 IU/ml penicillin and
additionally supplemented with 10 mg/ml insulin Sigma.
5.2.3.2: Cell inoculation
Adherent cells at a logarithmic growth phase were detached by addition of 2–3 ml
of a 0.05% trypsin (Gibco) 1– 0.02% EDTA mixture and incubated for 2–5 min at
37◦C. Cells were plated 200 ml per well in 96-well flat bottom microplates at densities
of 1,000–100,000 cells per well. Back- ground control wells (n = 8), containing the
same volume of complete culture medium, were included in each experiment. Cells
were plated in sextuplicate (six replicate wells per cell density) and experiments were
performed twice. Microplates were left for 6 h at 37◦C so that cells were able to attach
to the bottom of the wells before the fixation protocol was carried out.
5.2.3.3: Fixation protocol
In this method (Classic fixation protocol) (Skehan et al., 1990; Skehan, 1995),
50 µl of cold (4◦C) 50% TCA were added to the top of 200 µl culture medium in each
well to produce a final TCA concentration of 10%. TCA was gently layered (using an
8-channel, 50 µl multipipette, tips touching the culture medium surface at the edge of
the well) to avoid fluid shearing forces which could result in cell detachment and loss.
Microplates were left for 30 min at 4◦C and subsequently washed 5 times with
deionized water. Microplates were then dried with blow dryer.
5.2.4: Sulforhodamine B (SRB) antiproliferative assay
The SRB assay was carried out as previously described (Skehan et al., 1990;
Skehan, 1995). 70 µl 0.4% (w/v) Sulforhodamine B (Sigma) in 1% acetic acid
solution were added to each well and left at room temperature for 20 min. SRB was
removed and the plates washed 5 times with 1% acetic acid before air drying. Bound
SRB was solubilized with 200 µl 10 mM unbuffered Tris-base solution (Sigma) and
plates were left on a plate shaker for at least 30 min. Absorbance was read in a 96well plate reader (Anthos-2001, Anthos labteck instruments, A-5022, Salzburg) at 492
nm subtracting the background measurement at 620 nm. The test optical density (OD)
value was defined as the absorbance of each individual well, minus the blank value
(‗blank‘ is the mean optical density of the background control wells, n = 8). Mean
values and CV from six replicate wells were calculated automatically. Curves and
statistical analysis were performed using Excel 7.0 software.
IC50 values were derived by using curve-fitting methods with statistical analysis
software or IC50 calculation software.
Percentage of control cell growth =
Mean OD sample – Mean OD day 0
* 100
Mean OD negative control - mean OD day 0
Percentage growth inhibition = 100 - Percentage of control cell growth
5.3: Results
5.3.1: Analysis of transformed and untransformed Artemisia annua and
Artemisia dubia on breast cancer cell lines
Transformed and Un-transformed plants of Artemisia annua and Artemisia
dubia and hairy roots from these plants were analyzed to check their affectivity
against cancer by using MCF-7 breast cancer cell lines. Plants were analyzed for
anticancer activity by using method (described in materials and methods (Section:
5.2.4)) of Vanicha and Kanyawim (2006) Sulforhodamine B (SRB) antiproliferative
assay.
Sulforhodamine B (SRB) antiproliferative assay was used to test anticancer
activity of aqueous and hexane extracts of all transformed and untransformed plants
and hairy roots of Artemisia annua and Artemisia dubia against MCF7 breast cancer
cell lines at 50 µg concentration initially. The samples which showed more than 50%
inhibition at 50 µg were tested further at lower concentrations i.e. at 1.0 µg and 0.1 µg
to find out their IC50 values. The percentage of proliferation inhibition from transgenic
lines of Artemisia annua and Artemisia dubia varied between tissues, the difference
between the lines is shown (Fig. 5.1 A, B, C). The results of SRB assay showed that
hexane fractions of both transformed plants of Artemisia annua and Artemisia dubia
showed better proliferation inhibition than aqueous fractions against breast cancer cell
lines because artemisinin is better dissolved in hexane than water which confirmed the
effect of artemisinin on cancer cells compared to other secondary metabolites present
in transformed and untransformed Artemisia annua and Artemisia dubia. However,
the possibility of other effective compounds present in hexane extraction cannot be
excluded.
The hexane fractions of transformed plants of Artemisia annua showed 98%
proliferation inhibition compared to 55% proliferation inhibition in the controls
plants. Hairy roots of Artemisia annua also showed significantly greater (91-93%)
proliferation inhibition compared to control roots in which proliferation inhibition was
51%. The hexane fraction of transformed plants of Artemisia dubia showed lower
percentage of proliferation inhibition than A.annua, this was in transformed plants
93% compared to control plants 49%. Hairy roots of Artemisia dubia also showed
significantly higher proliferation inhibition (85%) compared to control roots (47%)
(Fig 5.1A).
The aqueous fractions of transformed plants of Artemisia annua and Artemisia
dubia showed lower activity as compared to hexane fractions. In aqueous fractions
again transformed plants and hairy roots of Artemisia annua showed higher
proliferation inhibition compared to transgenic plants of Artemisia dubia (Fig. 5.1B).
The aqueous fractions of transformed plants of Artemisia annua showed 89%
proliferation inhibition compared to 45% proliferation inhibition in the controls
plants. Hairy roots of Artemisia annua showed significantly higher (82-84%)
proliferation inhibition compared to control roots in which proliferation inhibition was
40%. The aqueous fraction of transformed plants of Artemisia dubia showed lower
percentage of proliferation inhibition than A.annua, this was higher in transformed
plants (84%) compared to control plants (43%). Hairy roots of Artemisia dubia also
showed significantly higher proliferation inhibition (75%) compared to control roots
which showed negligible proliferation inhibition (Fig 5.1B).
Overall results of IC50 values showed that hexane fraction of transformed and
untransformed plants of Artemisia annua and Artemisia dubia had the lowest IC50
values compared to aqueous fraction of transformed and untransformed plants of
Artemisia annua and Artemisia dubia against tested MCF7 breast cancer cell lines
(Fig. 5.1) because artemisinin is better dissolved in hexane than water (Lapkin and
Plucinski, 2006; Wright et al., 2010 ) which confirmed the effect of artemisinin on
cancer cells compared to other secondary metabolites present in transformed and
untransformed Artemisia annua and Artemisia dubia. As shown in the fig 5.2,
artemisinin content and anticancer activity were significantly correlated with each
other in samples of hexane extracts compared to aqueous extracts. Increased
anticancerous activity can also be related with the enhanced production of various
secondary metabolites including artemisinin in rol genes transformed plants.
Percentage Survival at 50 µg
60
50
90.01
73.6
66.6
51.41
40
30
4.71
20
2.27
10
1.3
1.75
1.84
2.43
2.01
2.53
3.03
1.96
0
Fig. 5.1 (A): Analysis of percentage survival in hexane fractions of transformed
and untransformed plants, roots and hairy roots of A.annua and A.dubia at 50 µg
concentration. The IC50 values are shown as values above the bars.
*Artemisinin (IC 50 = 0.21 µM) was used as positive control
140
330
Percentage Suvival at 50 µg
120
100
80
164
109
60
121.5
40
20
9.6
3
3.06
3.9
4.05
4.5
4.01
3.5
4.95
6.04
0
Fig. 5.1 (B): Analysis of percentage survival in aqueous fractions of transformed
and untransformed plants, roots and hairy roots of A.annua and A.dubia at 50 µg
concentration. The IC50 values are shown as values above the bars.
*Artemisinin (IC 50 = 0.21 µM) was used as positive control
Hexane Fraction
120.00
Percentage Inhibition at 50 µg
Aqueous Fraction
100.00
1.63
2.16
2.35
3.32
4.27
4.15
3.87
80.00
60.00
51.41
109
7.82
66.6
73.6
90.01
164
121.5
40.00
330
20.00
0.00
Fig. 5.1 (C): Comparative analysis of average percentage inhibition in hexane
and aqueous fractions of transformed and untransformed plants, roots and hairy
roots of A.annua and A.dubia at 50 µg concentration. The IC50 values are shown
as values above the bars.
*Artemisinin (IC 50 = 0.21 µM) was used as positive control
R-Squared Values
1.20
1.00
0.80
Aqeous Extraction
Hexane Extraction
0.99
0.99
0.99
0.74
0.74
0.70
0.68
0.62
0.60
0.48
0.52
0.51
0.43
0.66
0.56
0.43
0.40
0.23
0.20
0.00
Fig. 5.2: Comparative correlation analysis between artemisinin content and
anticancer activity in hexane and aqueous fractions of transformed and
untransformed plants, roots and hairy roots of A.annua and A.dubia. The R2
values are shown as values above the bars. Those samples that had R2 value 0.60.9 were significantly correlated. Data was analyzed for significant correlation by
using Graph Pad Prism Method.
5.4: Conclusion
In this part, hexane and aqueous extracts from transformed and
untransformed plants of Artemisia annua and Artemisia dubia were analyzed for
anticancerous activity against MCF-7 breast cancer cell lines and following
conclusion were drawn:
Crude extracts of rol genes transgenic plants revealed higher anticancerous
activities against MCF-7 breast cancer cell compared to control plants.
Hexane extracts of rol ABC genes transgenic plants revealed higher
anticancerous activity against MCF-7 breast cancer cell lines compared to
aqueous extracts of plants indicating may be this activity was due to
enhanced amount of artemisinin in hexane solvent.
Increased anticancerous activity could be related with the enhanced
production of other secondary metabolites including artemisinin in rol
transformed plants.
Chapter No. 6
Sequence analysis of Artemisia annua and Artemisia dubia and comparison in
transformed and untransformed plants
6.1: Introduction
Plants comprise some 400,000 species and are tremendously diverse in growth
habit, environmental adaptation, and nuclear genome structure. Plant genomes tend to
be large and complex, varying in size from ~38 Mb (1C) for the crucifer Cardamine
amara to > 87,000 Mb for Fritillaria assyriaca, a member of the Lilliaceae (Flavell et
al., 1974; Bennett and Leitch, 1995). Despite this diversity, plant geneticists have
found that plants exhibit extensive conservation of both gene content and gene order
(Bennetzen and Freeling, 1993). Since then comparative genetic analyses have begun
to show that different plant species often use homologous genes for very similar
functions (Fatokun et al., 1992; Ahn et al., 1993; Paterson et al., 1995; Lagercrantz et
al., 1996).
Different species evolved as a result of speciation event during the course of
evolution (John and Jeroen, 2004; Ohta, 1989). We come across increasingly complex
genomes as we move up the evolutionary tree. There can be several reasons that have
caused the complexity of gene number. Genes can be acquired from other species or
they can be duplicated from the existing genes. Duplication of single gene or group of
genes (Ohta, 2000), entire genome or a single chromosome or part of it can result in
large genome sequences. Sequences that emerged as a result of recombination within
the genome or were carried across the species by any means lead to divergence of
genomes (Posada et al., 2002). Replication slippage and DNA amplification (Romero
and Palacios, 1997) mostly create gene duplication. These evolutionary tools lead to
sequence homology amongst different organisms. Their genomes overlap to varying
degree depending upon the time of divergence.
In this study, genome of A.annua and A.dubia was sequenced for the first time.
Different contigs were generated by alignment of different samples individually from
both A.annua and A.dubia to evaluate whether genes in the genome of transformed
plants are up or down regulated compared to untransformed plants. This up and down
regulation comparison was also made between A.annua and A.dubia. Furthermore,
genome sequences of individual transgenic lines were also compared. BLAST of
these contigs was performed using nr/nt database in NCBI. The homology of their
genome sequence was found and significant results were obtained.
6.2: Materials and Methods
6.2.1: Sequence analysis of transformed and untransformed A.annua and
A.dubia
Sequence analysis of transformed and untransformed plants of Artemisia annua
and Artemisia dubia was performed to find out differences in sequences of
transformed and untransformed plants.
To make libraries, 4µg of total RNA (RNA was extracted according to method
described in section 4.2.1.1) was isolated and then sheared using a chemical reaction.
This material was then used to create the libraries. The whole protocol is described
below.
6.2.2: Purification and fragmentation of mRNA
This process purifies the poly‐A containing mRNA molecules using poly‐T
oligo‐attached magnetic beads using two rounds of purification. During the second
elution of the poly‐A RNA, the RNA is also fragmented and primed for cDNA
synthesis.
6.2.2.1: Formation and purification of RNA bead plate
Following protocol was used to make and purify RBP
4µg of total RNA was diluted with nuclease‐free ultra pure water to a final
volume of 50 μl in a new 96‐well 0.3 ml PCR plate with the RBP barcode
label.
Thawed RNA Purification Beads tube was vigorously vortexed to completely
resuspend the oligo‐dT beads.
50 μl of RNA Purification Beads were added to each well of the RBP plate
using a multichannel pipette to bind the poly‐A RNA to the oligo dT magnetic
beads. The entire volume was gently pipetted up and down 6 times to mix
thoroughly. The tips were changed after each column.
RBP plate was then sealed with a Microseal ‗B‘ Adhesive seal.
Sealed RBP plate was placed on the pre-programmed thermal cycler. The lid
was closed and mRNA Denaturation was selected (65• ◦C for 5 minutes, 4◦C
hold) to denature the RNA and facilitate binding of the poly-A RNA to the
beads.
RBP plate was removed from the thermal cycler when it reached 4 ◦C.
RBP plate was placed on the bench and incubated at room temperature for 5
minutes to allow the RNA to bind to the beads.
RBP plate was placed on the magnetic stand at room temperature for 5
minutes to separate the poly -A RNA bound beads from the solution.
Adhesive seal was removed from the RBP plate.
All of the supernatant was removed and discarded from each well of the RBP
plate using a multichannel pipette. Tips were changed after each column.
RBP plate was removed from the magnetic stand.
Beads were washed by adding 200 μl of Bead Washing Buffer in each well of
the RBP plate using a multichannel pipette to remove unbound RNA. The
entire volume was gently pipetted up and down 6 times to mix thoroughly.
The tips were changed after each column.
RBP plate was placed on the magnetic stand at room temperature for 5
minutes.
Elution Buffer was thawed and centrifuged to 600 xg for 5 seconds.
All of the supernatant was removed and discarded from each well of the RBP
plate using a multichannel pipette. The tips were changed after each column.
RBP plate was removed from the magnetic stand.
50 μl of Elution Buffer was added in each well of the RBP plate using a
multichannel pipette. The entire volume was gently pipetted up and down 6
times to mix thoroughly. The RBP plate was sealed with a Microseal B
Adhesive seal. The tips were changed after each column.
Elution Buffer tube was stored at 4°C.
Sealed RBP plate was placed on the pre‐programmed thermal cycler. Lid was
closed and mRNA Elution 1 was selected (80°C for 2 minutes, 25°C hold) to
elute the mRNA from the beads.
RBP plate was removed from the thermal cycler when it reached 25°C.
RBP plate was placed on the bench at room temperature and adhesive seal was
removed from the plate.
6.2.2.2: Formation and purification of RNA fragmentation plate
Following protocol was used to make and purify RFP
50 µl of Bead Binding Buffer was added to each well of the RBP plate using a
multichannel pipette to allow the RNA to re-bind to the beads. The entire
volume was gently pipetted up and down 6 times to mix thoroughly. The tips
were changed after each column.
RBP plate was incubated at room temperature for 5 minutes and the Bead
Binding Buffer tube was stored at 2°C to 8°C.
RBP plate was placed on the magnetic stand at room temperature for 5
minutes.
All of the supernatant was removed and discarded from each well of the RBP
plate using a multichannel pipette. Tips were changed after each column.
RBP plate was removed from the magnetic stand.
Beads were washed by adding 200 µl of Bead Washing Buffer in each well of
the RBP plate using a multichannel pipette. The entire volume was gently
pipetted up and down 6 times to mix thoroughly. The tips were changed after
each column.
Bead Washing Buffer tube was stored with at 2°C to 8°C.
RBP plate was placed on the magnetic stand at room temperature for 5
minutes.
All of the supernatant was removed and discarded from each well of the RBP
plate using a multichannel pipette. Tips were changed after each column.
RBP plate was removed from the magnetic stand.
19.5 µl of Elute was added, Primed, and Fragmented and mixed to each well
of the RBP plate using a multichannel pipette. The entire volume was gently
pipetted up and down 6 times to mix thoroughly. The tips were changed after
each column. The Elute, Prime, Fragment Mix contains random hexamers for
RT priming and serves as the 1st strand cDNA synthesis reaction buffer.
RBP plate was sealed with a Microseal ‗B‘ Adhesive seal.
Elute, Prime, Fragment Mix tube was stored at ‐15° to ‐25°C.
Sealed RBP plate was placed on the pre‐programmed thermal cycler. Lid was
closed and Elution 2 ‐ Frag ‐ Prime was selected (94°C for 8 minutes, 4°C
hold) to elute, fragment, and prime the RNA.
RBP plate was removed from the thermal cycler when it reaches 4°C and
centrifuged briefly.
6.2.3: Synthesis of first strand cDNA
This process reverse transcribes the cleaved RNA fragments primed
with random hexamers into first strand cDNA using reverse transcriptase and
random primers.
6.2.3.1: Formation of cDNA plate
RBP plate was placed on the magnetic stand at room temperature for 5
minutes.
Adhesive seal was removed from the RBP plate.
17 μl of the supernatant (fragmented and primed mRNA) was transferred from
each well of the RBP plate to the corresponding well of the new 0.3 ml PCR
plate labeled with the CDP barcode. Some liquid may remain in each well.
The thawed First Strand Master Mix was centrifuged to 600 xg for 5 seconds.
50 μl SuperScript II First Strand Master Mix tube (ratio: 1 μl SuperScript II
for each 7 μl First Strand Master Mix). Mixed gently, but thoroughly, and
centrifuged briefly.
First Strand Master Mix tube was labeled to indicate that the SuperScript II
has been added.
8 μl of First Strand Master Mix and SuperScript II mix was added to each well
of the CDP plate using a multichannel pipette. The entire volume was gently
pipetted up and down 6 times to mix thoroughly. The tips were changed after
each column.
CDP plate was sealed with a Microseal ‗B‘ Adhesive seal and centrifuge
briefly.
First Strand Master Mix tube back was returned to ‐15° to ‐25°C storage
immediately after use.
CDP plate was incubated on the thermal cycler, with the lid closed, using the
1st Strand program:
25°C for 10 minutes
42°C for 50 minutes
70°C for 15 minutes
Hold at 4°C
When the thermal cycler reached 4°C, the CDP plate was removed from the
thermal cycler.
6.2.4: Synthesis of second strand cDNA
6.2.4.1: Addition of second strand master mix
The thawed Second Strand Master Mix was centrifuged to 600 xg for 5
seconds.
Adhesive seal was removed from the CDP plate.
25 μl of thawed Second Strand Master Mix was added to each well of the CDP
plate using a multichannel pipette. The entire volume was gently pipetted up
and down 6 times to mix thoroughly. The tips were changed after each
column.
CDP plate was sealed with a Microseal ‗B‘ Adhesive seal.
CDP plate was incubated on the pre‐heated thermal cycler; lid was closed and
placed at 16°C for 1 hour.
CDP plate was removed from the thermal cycler; adhesive seal was removed,
and placed the plate to room temperature.
AMPure XP beads were vortexed until they were dispersed, then 90 μl of
well‐mixed AMPure XP beads were added to each well of the CDP plate
containing 50 μl of ds cDNA. The entire volume was gently pipetted up and
down 10 times to mix thoroughly.
CDP plate was incubated at room temperature for 15 minutes.
CDP plate was placed on the magnetic stand at room temperature, for 5
minutes to ensure that all of the beads were bounded to the side of the wells.
135 μl of the supernatant was removed and discarded from each well of the
CDP plate using a multichannel pipette. The tips were changed after each
column.
200 μl of freshly prepared 80% EtOH was added to each well without
disturbing the beads.
CDP plate was incubated at room temperature for 30 seconds, and then all of
the supernatant was removed and discarded from each well using a
multichannel pipette. The tips were changed after each column.
Steps 5 and 6 were repeated once for a total of two 80% EtOH washes.
Plate was placed at room temperature for 15 minutes to dry and then removed
the CDP plate from the magnetic stand.
The thawed, room temperature Resuspension Buffer was centrifuged to 600 xg
for 5 seconds.
52.5 µl OF Resuspension Buffer was added to each well of the CDP plate
using a multichannel pipette. The entire volume was gently pipetted up and
down 10 times to mix thoroughly.
CDP plate was incubated at room temperature for 2 minutes.
CDP plate was placed on the magnetic stand at room temperature for 5
minutes.
50 µl of the supernatant (ds cDNA) was transferred from the CDP plate to the
new 0.3 ml PCR plate labeled with the IMP barcode.
6.2.5: End repairing
This process converts the overhangs resulting from fragmentation into blunt
ends, using an End Repair (ERP) mix. The 3' to 5' exonuclease activity of this mix
removes the 3' overhangs and the polymerase activity fills in the 5' overhangs.
6.2.5.1: Formation of insert modification plate
The thawed End Repair Control tube was centrifuged to 600 xg for 5 seconds
and the End Repair Control was diluted to 1/100 in Resuspension Buffer (1 μl
End Repair Control + 99 μl Resuspension Buffer) before use. The diluted End
Repair Control was discarded after use.
10 μl of diluted End Repair Control (or 10 μl of Resuspension Buffer if not
using End Repair Control) was added to each well of the IMP plate that
contains 50 μl of ds cDNA using a multichannel pipette. Tips were changed
after each column.
40 μl of End Repair Mix was added to each well of the IMP plate containing
the ds cDNA and End Repair Control using a multichannel pipette. The entire
volume was gently pipetted up and down 10 times to mix thoroughly.
IMP plate was sealed with a Microseal ‗B‘ adhesive seal.
IMP plate was incubated on the pre‐heated thermal cycler, with the lid closed,
at 30°C for 30 minutes.
IMP plate was removed from the thermal cycler.
Adhesive seal was removed from the IMP plate.
AMPure XP Beads were vortexed until they were well dispersed and 160 μl of
well‐mixed AMPure XP Beads was added to each well of the IMP plate
containing 100 μl of End Repair Mix. It was gently pipetted up and down 10
times to mix thoroughly.
IMP plate was incubated at room temperature for 15 minutes.
IMP plate was placed on the magnetic stand at room temperature for at least 5
minutes, until the liquid appears clear.
With 200 μl multichannel pipett set to 127.5 μl, 127.5 μl of the supernatant
was removed from each well of the IMP plate. Care was taken not to disturb
the beads. The tips were changed after each column.
Step 5 was repeated once.
200 μl of freshly prepared 80% EtOH was added to each well without
disturbing the beads.
IMP plate was incubated at room temperature for at least 30 seconds, then
removed and discarded all of the supernatant from each well. Care was taken
not to disturb the beads. The tips were changed after each column.
Steps 7 and 8 were repeated once for a total of two 80% EtOH washes.
IMP plate was placed at room temperature for 15 minutes to dry and then plate
was removed from the magnetic stand.
Dried pellet was resuspended in 17.5 μl Resuspension Buffer. The entire
volume was gently pipetted up and down 10 times to mix thoroughly.
IMP plate was incubated at room temperature for 2 minutes.
IMP plate was placed on the magnetic stand at room temperature for at least 5
minutes, until the liquid appeared clear.
15 μl of the clear supernatant was transferred from each well of the IMP plate
to the corresponding well of the new 0.3 ml PCR plate labeled with the ALP
barcode.
6.2.6: 3' Ends adenylation
A single ‗A‘ nucleotide is added to the 3‘ ends of the blunt fragments to prevent
them from ligating to one another during the adapter ligation reaction. A
corresponding single ‗T‘ nucleotide on the 3‘ end of the adapter provides a
complementary overhang for ligating the adapter to the fragment. This strategy
ensures a low rate of chimera (concatenated template) formation.
6.2.6.1: Addition of A-Tailing mix
The thawed A-Tailing Control tube was centrifuged to 600 xg for 5 seconds
and the A-Tailing Control was diluted to 1/100 in Resuspension Buffer (1 µl
A-Tailing Control + 99 µl Resuspension Buffer) before use. The diluted ATailing Control was discarded after use.
2.5 µl of diluted A-Tailing Control (or 2.5 µl of Resuspension Buffer if not
using A-Tailing Control) was added to each well of the ALP plate using a
multichannel pipette.
Multichannel pipette was adjusted to 30 µl and the entire volume was gently
pipetted up and down 10 times to mix thoroughly.
12.5 µl of thawed A-Tailing Mix was added to each well of the ALP plate
using a multichannel pipette. Tips were changed after each column.
ALP plate was sealed with a Microseal B adhesive seal.
ALP plate was incubated on the pre-heated thermal cycler, with the lid closed,
at 37°C for 30 minutes.
After 30 minutes ALP plate was removed from the thermal cycler.
6.2.7: Ligation of adapters
This process ligates multiple indexing adapters to the ends of the ds cDNA,
preparing them for hybridization onto a flow cell.
6.2.7.1: Addition of ligation mix
Thawed RNA Adapter Index tubes (AR001–AR012 depending on the
RNA Adapter Indexes being used), Ligase Control, and Stop Ligase Mix
tubes were centrifuged to 600 xg for 5 seconds.
DNA Ligase Mix tube was removed immediately from ‐15° to ‐25°C
storage before use.
Adhesive seal was removed from the ALP plate.
2.5 µl of DNA Ligase Mix was added to each well of the ALP plate.
DNA Ligase Mix tube was returned back to 15 ° to 25 °C storage
immediately after use.
Ligase Control was diluted to 1/100 in Resuspension Buffer (1 µl Ligase
Control + 99 µl Resuspension Buffer) before use. The diluted Ligase
Control was discarded after use.
2.5 μl of diluted Ligase Control (or 2.5 μl of Resuspension Buffer if not
using Ligase Control) was added to each well of the ALP plate.
2.5 μl of each thawed RNA Adapter Index (AR001–AR012 depending on
the RNA Adapter Indexes being used) was added to each well of the ALP
plate using a multichannel pipette.
Multichannel pipette was adjusted to 40 μl and the entire volume was
gently pipetted up and down 10 times to mix thoroughly.
ALP plate was sealed with a Microseal ‗B‘ adhesive seal.
ALP plate was incubated on the pre‐heated thermal cycler, with the lid
closed, at 30°C for 10 minutes.
ALP plate was removed from the thermal cycler and Adhesive seal was
removed from the ALP plate.
5 μl of Stop Ligase Mix was added to each well of the ALP plate to
inactivate the ligation mix using a multichannel pipette. The entire volume
was gently pipetted up and down 10 times to mix thoroughly.
AMPure XP Beads were vortexed until they are well dispersed, then 42 μl
of mixed AMPure XP Beads was added to each well of the ALP plate
using a multichannel pipette. The entire volume was gently pipetted up and
down 10 times to mix thoroughly.
ALP plate was placed at room temperature for 15 minutes.
ALP plate was incubated on the magnetic stand at room temperature for at
least 5 minutes, until the liquid appears clear.
79.5 μl of the supernatant from each well of the ALP plate was removed
and discarded using a multichannel pipette. Some liquid may remain in
each well. Tips were changed after each column.
200 μl of freshly prepared 80% EtOH was added to each well without
disturbing the beads.
ALP plate was incubated at room temperature for at least 30 seconds, and
then all of the supernatant was removed and discarded from each well.
Care was taken not to disturb the beads. The tips were changed after each
column.
Steps 5 and 6 were repeated once for a total of two 80% EtOH washes.
ALP plate was placed at room temperature for 15 minutes to dry and then
plate was removed from the magnetic stand.
Dried pellet was resuspended in each well with 52.5 μl Resuspension
Buffer. Gently pipetted the entire volume up and down 10 times to mix
thoroughly.
ALP plate was incubated at room temperature for 2 minutes.
ALP plate was placed on the magnetic stand at room temperature for at
least 5 minutes, until the liquid appeared clear.
50 μl of the clear supernatant was transferred from each well of the ALP
plate to the corresponding well of the new 0.3 ml PCR plate labelled with
the CAP barcode. Some liquid may remain in each well. Tips were
changed after each column.
AMPure XP Beads were vortexed until they were well dispersed, then 50
μl of mixed AMPure XP Beads was added to each well of the CAP plate
for a second clean up using a multichannel pipette. The entire volume was
gently pipetted up and down 10 times to mix thoroughly.
CAP plate was incubated at room temperature for 15 minutes and then on
the magnetic stand at room temperature for 5 minutes or until the liquid
appears clear.
95 μl of the supernatant was removed and discarded from each well of the
CAP plate, using a multichannel pipette. The tips were changed after each
column.
200 μl of freshly prepared 80% EtOH with the CAP plate remaining on the
magnetic stand was added to each well without disturbing the beads.
CAP plate was incubated at room temperature for at least 30 seconds, and
then all of the supernatant was removed and discarded from each well.
Care was taken not to disturb the beads. The tips were changed after each
column.
Steps 17 and 18 were repeated once for a total of two 80% EtOH washes.
CAP plate was placed at room temperature for 15 minutes to dry and then
removed the plate from the magnetic stand.
Dried pellet was resuspended in each well with 22.5 μl Resuspension
Buffer. The entire volume was gently pipetted up and down 10 times to
mix thoroughly.
CAP plate was incubated at room temperature for 2 minutes.
CAP plate was placed on the magnetic stand at room temperature for at
least 5 minutes, until the liquid appeared clear.
20 μl of the clear supernatant was transferred from each well of the CAP
plate to the corresponding well of the new 0.3 ml PCR plate labeled with
the PCR barcode.
6.2.8: DNA fragments enrichment
This process uses PCR to selectively enrich those DNA fragments that have
adapter molecules on both ends and to amplify the amount of DNA in the library.
The PCR is performed with a PCR primer cocktail that anneals to the ends of the
adapters. The number of PCR cycles should be minimized to avoid skewing the
representation of the library.
6.2.8.1: PCR reaction
5 µl of thawed PCR Primer Cocktail was added to each well of the PCR
plate using a multichannel pipette. The tips were changed after each
column.
25 µl of thawed PCR Master Mix was added to each well of the PCR plate
using a multichannel pipette. The tips were changed after each column.
Single channel or multichannel pipette was adjusted to 40 µl and the entire
volume was gently pipetted up and down 10 times to mix thoroughly.
6.2.8.2: Amplification of PCR plates
PCR plate was amplified in the pre-programmed thermal cycler, with the
lid closed, using the PCR program:
98°C for 30 seconds
15 cycles of:
98°C for 10 seconds
60°C for 30 seconds
72°C for 30 seconds
72°C for 5 minutes
Hold at 4°C
6.2.8.3: Cleaning of PCR plates
AMPure XP Beads were vortexed until they were well dispersed, 50 µl of the
mixed AMPure XP Beads was added to each well of the PCR plate containing
50 µl of the PCR amplified library using a multichannel pipette. The entire
volume was gently pipetted up and down 10 times to mix thoroughly.
PCR plate was incubated at room temperature for 15 minutes.
PCR plate was placed on the magnetic stand at room temperature for at least 5
minutes, until the liquid appeared clear.
95 μl of the supernatant was removed and discarded from each well of the
PCR plate, using a multichannel pipette. The tips were changed after each
column.
200 μl of freshly prepared 80% EtOH was added to each well without
disturbing the beads.
PCR plate was incubated at room temperature for at least 30 seconds, and then
all of the supernatant was removed and discarded from each well. The tips
were changed after each column.
Steps 5 and 6 were repeated once for a total of two 80% EtOH washes.
PCR plate was placed at room temperature for 15 minutes to dry and then
removed the plate from the magnetic stand.
Dried pellet was resuspended in each well with 32.5 μl Resuspension Buffer
using a multichannel pipette. The entire volume was gently pipetted up and
down 10 times to mix thoroughly.
PCR plate was incubated at room temperature for 2 minutes.
PCR plate was placed on the magnetic stand at room temperature for at least 5
minutes, until the liquid appeared clear.
30 μl of the clear supernatant was transferred from each well of the PCR plate
to the corresponding well of the new 0.3 ml PCR plate labeled with the TSP1
barcode. The tips were changed after each column.
6.2.9: Validation of library
6.2.9.1: Quantification of libraries
In order to achieve the highest quality of data on Illumina sequencing
platforms, it is important to create optimum cluster densities across every lane of
every flow cell. This requires accurate Quantitation of DNA library templates.
6.2.9.2: Quality control
1 μl of the resuspended construct was loaded on an Agilent Technologies 2100
Bioanalyzer using a DNA specific chip such the Agilent DNA‐1000.
Size and purity of sample was checked. The final product should be a band at
approximately 260 bp (for single‐read libraries).
6.3: Evaluation of sequence homology
Different contigs were generated by alignment of different samples
individually from both A.annua and A.dubia to evaluate whether genes in the genome
of transformed plants are up or down regulated compared to untransformed plants.
This up and down regulation comparison was also made between A.annua and
A.dubia. BLAST of these contigs was performed using nr/nt database. The homology
of their genome sequence was found and significant results were obtained. Putative
functions of genes showing homology were sorted using Expert Protein Analysis
resources.
6.4: Results
In this study, the transcriptome of Artemisia annua and Artemisia dubia were
sequenced for the first time. Gene contigs were generated by alignment of the
sequences derived from a compilation of all the sequences obtained from a species.
The different individual samples from both Artemisia annua and Artemisia dubia
were then compared against this reference gene list to evaluate if any genes in the
genome of the transformed plants are up or down regulated compared to
untransformed plants. The Bowtie and CuffLinks programs were used to look for
differential expression and CuffDiff used to derive values. A comparison was also
made between Artemisia annua and Artemisia dubia (Table 6.1) plants. Contigs/genes
for which significant differences were obtained were compared between biological
replicates to determine their overall significance. Those which showed significant
differences were submitted to the BLAST program using both the nr/nt and EST
database available at NCBI. The homology of their genome sequence was found and
significant results were obtained. From the results obtained the putative gene
functions could be derived. Furthermore, genome sequences of individual transgenic
lines, which were different from each other in copy number, were also compared.
Besides, genome sequence of untransformed and transformed Artemisia annua was
compared individually with genome sequence of both untransformed and transformed
Artemisia dubia
6.4.1: Comparison of various transgenic lines with respect to specific contigs
Transcriptome sequencing of individual A.annua and A.dubia plants allowed
transcript abundance to be measured. It permitted us to determine the genes which are
up or down regulated in response to transformation with the rol genes and also to
determine the likelihood that they are involved directly in artemisinin production.
Genes that were significantly upregulated in multiple plants compared to the controls
allowed a degree of significance to be added. Comparison between A.annua and
A.dubia also adds to the degree of certainty that these genes are important.
As shown in Table 6.1, contigs were generated by alignment of samples in
column sample_1 with samples in column sample_2 and significant value was
recorded. Significant values provided information about up and down regulation of
genes of respective samples. Those genes that had significant value less than 0.01
were up regulated.
Genes of different transgenic lines (A1 and A2) of A.annua were highly up
regulated as compared to the control plants (AC). Similar results were observed for
transgenic (D1) and control (DC) lines of A. dubia (Table 6.1). When transgenic lines
A1 and A2 of A.annua were compared with the transgenic line D1 of A. dubia, genes
of A1 and A2 showed up regulation. Transgenic lines of A.annua were analyzed
against control plants of A.dubia and genes of A.annua were found upregulated.
Interesting results were seen when control lines of A. annua showed up regulation in
comparison with transgenic lines of A. dubia.
In this study, total number of genes identified as upregulated in each
comparison produced a huge data. However, in each case, those genes that showed
more than 90% homology have been shown (Table 6.1 and 6.2). Samples in column
Sample_1 showed up regulation of genes as compared to the samples in column
Sample_2 (Table 6.1). Number of genes that maintain 90% homology vary in each
case. When A1 transgenic line was compared with A2, five genes were found
upregulated. Comparison of A1 transgenic line with AC, A1 with D1, A2 with AC,
and A2 with D1 showed 67, 72, 81, 63 genes upregulated respectively.
50 genes were found upregulated in comparison of the AC with D1, 28 genes
of A1 with DC, 30 in A2 with DC, 55 in AC and DC and 29 genes were found
upregulated in comparison of D1 with DC (Table 6.1). In some cases whole genome
was found matching with the transcriptomic sequence of A.annua and A.dubia (Table
6.2).
Table 6.1: Comparison of various Transgenic Lines with respect to specific Contigs
S.No.
Contigs
Sample_1
Sample_2
Significant value
1
Contig3276:0-518
A1.1
A2.1
0.007521
2
Contig5332:0-407
A1.1
A2.1
0.00926317
3
Contig5575:0-732
A1.1
A2.1
0.0263234
4
Contig57:9-676
A1.1
A2.1
0.00230373
5
Contig9512:0-1058
A1.2
A2.1
0.0152992
6
Contig5987:0-452
A1.1
A2.2
0.00645269
7
Contig6284:0-818
A1.1
A2.2
2.93E-05
8
Contig9144:0-754
A1.1
A2.2
9.52E-07
9
Contig951:0-251
A1.1
A2.2
0.0199822
10
Contig100:43-844
A1.1
AC.1
8.10E-05
11
Contig10524:0-757
A1.1
AC.1
0.000706668
12
Contig10536:0-983
A1.1
AC.1
0.00529889
13
Contig1181:0-4663
A1.1
AC.1
0.00408348
14
Contig1260:39-212
A1.1
AC.1
0.00822729
15
Contig1320:0-1308
A1.1
AC.1
0.0272819
16
Contig1606:21-1047
A1.1
AC.1
0.00080821
17
Contig161:0-979
A1.1
AC.1
4.06E-06
18
Contig167:6-215
A1.1
AC.1
0.0397222
19
Contig1746:0-2322
A1.1
AC.1
0.0184849
20
Contig2006:0-880
A1.1
AC.1
0.00328411
21
Contig2028:0-994
A1.1
AC.1
0.0489583
22
Contig2219:42-1241
A1.1
AC.1
0.00549141
23
Contig2799:0-245
A1.1
AC.1
0.00353468
24
Contig2903:0-2391
A1.1
AC.1
0.0170782
25
Contig8369:11-1513
A1.2
AC.1
4.68E-07
26
Contig2157:15-764
A1.1
AC.2
2.29E-07
27
Contig5597:0-1737
A1.1
AC.2
8.97E-08
28
Contig2380:0-1787
A1.2
AC.2
5.24E-12
29
Contig231:0-650
A1.2
AC.1
0.000630584
30
Contig249:21-956
A1.2
AC.1
0.000107155
31
Contig253:0-1100
A1.2
AC.1
0.000110396
32
Contig2799:0-245
A1.2
AC.1
0.000816532
33
Contig2859:34-864
A1.2
AC.1
0.000625534
34
Contig3121:0-262
A1.2
AC.1
0.00026362
35
Contig2157:15-764
A2.1
AC.2
2.90E-09
36
Contig8369:11-1513
A2.1
AC.1
5.05E-08
37
Contig2380:0-1787
A2.2
AC.2
1.90E-08
38
Contig5597:0-1737
A2.2
AC.2
4.96E-08
39
Contig10105:0-1419
A2.1
AC.2
6.57E-05
40
Contig161:0-979
A2.1
AC.2
9.64E-10
41
Contig3836:0-1711
A2.1
AC.2
5.10E-08
42
Contig3946:2-446
A2.1
AC.2
3.66E-08
43
Contig515:13-967
A2.1
AC.2
3.61E-11
44
Contig8590:0-1715
A2.1
AC.2
1.35E-08
45
Contig8696:0-1843
A2.1
AC.2
1.42E-08
46
Contig3836:0-1711
A2.2
AC.2
3.61E-11
47
Contig3840:0-1556
A2.2
AC.2
5.51E-08
48
Contig8590:0-1715
A2.2
AC.2
1.83E-08
49
Contig1972:29-1001
A2.1
AC.2
4.10E-05
50
Contig231:0-650
A2.1
AC.2
9.41E-06
51
Contig2622:14-797
A2.1
AC.2
3.38E-05
52
Contig3934:0-1618
A2.1
AC.2
9.68E-05
53
Contig4225:0-563
A2.1
AC.2
3.50E-05
54
Contig4679:38-421
A2.1
AC.2
1.59E-05
55
Contig5845:0-796
A2.1
AC.2
1.24E-05
56
Contig981:16-763
A2.1
AC.2
2.25E-05
57
Contig10105:0-1419
A2.2
AC.2
8.41E-05
58
Contig1907:54-728
A2.2
AC.2
3.83E-05
59
Contig231:0-650
A2.2
AC.2
9.02E-06
60
Contig3934:0-1618
A2.2
AC.2
3.74E-05
61
Contig4225:0-563
A2.2
AC.2
8.01E-05
62
Contig436:0-616
A2.2
AC.2
1.50E-06
63
Contig4679:38-421
A2.2
AC.2
1.50E-05
64
Contig4749:0-1057
A2.2
AC.2
3.82E-05
65
Contig8881:0-761
A2.2
AC.2
3.52E-06
66
Contig981:16-763
A2.2
AC.2
1.38E-05
67
Contig10105:0-1419
A1.1
D1.1
1.78E-06
68
Contig1388:5-1056
A1.1
D1.1
2.10E-05
69
Contig296:0-295
A1.1
D1.1
1.19E-06
70
Contig3057:0-1927
A1.1
D1.1
8.73E-12
71
Contig3437:0-1709
A1.1
D1.1
1.75E-10
72
Contig4685:0-615
A1.1
D1.1
5.65E-05
73
Contig5168:6-688
A1.1
D1.1
0.000201614
74
Contig5601:0-759
A1.1
D1.1
2.04E-07
75
Contig7008:0-1433
A1.1
D1.1
1.21E-06
76
Contig7150:0-1455
A1.1
D1.1
1.50E-05
77
Contig718:0-1984
A1.1
D1.1
2.01E-10
78
Contig7416:0-1030
A1.1
D1.1
0.000153826
79
Contig7540:0-1080
A1.1
D1.1
2.91E-05
80
Contig8220:0-844
A1.1
D1.1
1.59E-05
81
Contig10057:0-852
A1.2
D1.1
0.00151277
82
Contig10105:0-1419
A1.2
D1.1
2.23E-06
83
Contig1388:5-1056
A1.2
D1.1
1.70E-05
84
Contig1972:29-1001
A1.2
D1.1
4.96E-08
85
Contig209:0-247
A1.2
D1.1
0.000277453
86
Contig2454:22-1644
A1.2
D1.1
0
87
Contig249:21-956
A1.2
D1.1
0.000363911
88
Contig2799:0-245
A1.2
D1.1
0.000286909
89
Contig296:0-295
A1.2
D1.1
1.26E-06
90
Contig3057:0-1927
A1.2
D1.1
8.73E-12
91
Contig3437:0-1709
A1.2
D1.1
2.47E-10
92
Contig3946:2-446
A1.2
D1.1
8.57E-09
93
Contig5601:0-759
A1.2
D1.1
2.16E-07
94
Contig7008:0-1433
A1.2
D1.1
1.42E-06
95
Contig7150:0-1455
A1.2
D1.1
1.50E-05
96
Contig718:0-1984
A1.2
D1.1
1.50E-10
97
Contig7469:34-524
A1.2
D1.1
5.72E-05
98
Contig5597:0-1737
A1.2
D1.2
8.73E-12
99
Contig10057:0-852
A2.1
D1.1
3.16E-05
100
Contig10105:0-1419
A2.1
D1.1
2.46E-07
101
Contig1388:5-1056
A2.1
D1.1
1.88E-07
102
Contig1972:29-1001
A2.1
D1.1
4.96E-08
103
Contig2698:0-454
A2.1
D1.1
1.10E-07
104
Contig2867:23-797
A2.1
D1.1
1.10E-07
105
Contig296:0-295
A2.1
D1.1
5.46E-06
106
Contig3946:2-446
A2.1
D1.1
8.72E-09
107
Contig4155:0-1303
A2.1
D1.1
1.79E-05
108
Contig451:21-2271
A2.1
D1.1
2.16E-05
109
Contig4685:0-615
A2.1
D1.1
2.73E-05
110
Contig478:36-283
A2.1
D1.1
5.33E-05
111
Contig5575:0-732
A2.1
D1.1
2.52E-05
112
Contig5601:0-759
A2.1
D1.1
3.87E-09
113
Contig7008:0-1433
A2.1
D1.1
1.83E-08
114
Contig7015:0-567
A2.1
D1.1
7.88E-07
115
Contig7150:0-1455
A2.1
D1.1
1.33E-05
116
Contig718:0-1984
A2.1
D1.1
4.32E-11
117
Contig7219:0-736
A2.1
D1.1
2.93E-05
118
Contig10057:0-852
A2.2
D1.1
3.33E-05
119
Contig10105:0-1419
A2.2
D1.1
3.83E-07
120
Contig1388:5-1056
A2.2
D1.1
2.82E-07
121
Contig2698:0-454
A2.2
D1.1
1.07E-07
122
Contig2867:23-797
A2.2
D1.1
1.10E-07
123
Contig296:0-295
A2.2
D1.1
3.22E-06
124
Contig5601:0-759
A2.2
D1.1
2.90E-09
125
Contig5987:0-452
A2.2
D1.1
1.14E-08
126
Contig6284:0-818
A2.2
D1.1
1.71E-06
127
Contig718:0-1984
A2.2
D1.1
4.96E-11
128
Contig7219:0-736
A2.2
D1.1
3.19E-05
129
Contig5597:0-1737
A2.2
D1.2
5.24E-12
130
Contig5597:0-1737
A2.1
D1.2
0
131
Contig10461:0-582
AC.1
D1.1
4.88E-05
132
Contig2006:0-880
AC.1
D1.1
5.29E-07
133
Contig2126:0-1124
AC.1
D1.1
4.66E-11
134
Contig2288:0-726
AC.1
D1.1
1.20E-09
135
Contig231:0-650
AC.1
D1.1
4.40E-10
136
Contig2454:22-1644
AC.1
D1.1
4.48E-09
137
Contig2622:14-797
AC.1
D1.1
7.33E-06
138
Contig290:0-475
AC.1
D1.1
3.12E-09
139
Contig318:0-498
AC.1
D1.1
1.94E-05
140
Contig362:0-275
AC.1
D1.1
7.85E-06
141
Contig4168:0-828
AC.1
D1.1
1.71E-06
142
Contig418:2-2747
AC.1
D1.1
5.65E-05
143
Contig4225:0-563
AC.1
D1.1
1.66E-05
144
Contig4704:0-1313
AC.1
D1.1
2.65E-09
145
Contig5025:0-1950
AC.1
D1.1
6.92E-10
146
Contig5482:0-902
AC.1
D1.1
2.78E-07
147
Contig583:0-1445
AC.1
D1.1
3.77E-05
148
Contig6601:0-889
AC.1
D1.1
7.93E-07
149
Contig762:0-855
AC.1
D1.1
8.72E-07
150
Contig8568:0-1291
AC.1
D1.1
9.39E-05
151
Contig2380:0-1787
AC.2
D1.1
0
152
Contig4704:0-1313
AC.2
D1.1
3.26E-09
153
Contig4971:0-638
AC.2
D1.1
9.95E-07
154
Contig5008:0-2908
AC.2
D1.1
2.66E-08
155
Contig10081:0-821
A1.1
DC.1
3.40E-08
156
Contig10271:0-621
A1.1
DC.1
1.22E-05
157
Contig1852:0-2203
A1.1
DC.1
0
158
Contig2157:15-764
A1.1
DC.1
3.50E-08
159
Contig2380:0-1787
A1.1
DC.1
0.0092017
160
Contig3057:0-1927
A1.1
DC.1
2.19E-08
161
Contig5601:0-759
A1.1
DC.1
1.90E-05
162
Contig5821:0-949
A1.1
DC.1
5.32E-06
163
Contig7708:0-1368
A1.1
DC.1
4.88E-05
164
Contig9460:0-1091
A1.1
DC.1
8.06E-05
165
Contig9658:53-1094
A1.1
DC.1
5.85E-05
166
Contig10081:0-821
A1.2
DC.1
1.90E-08
167
Contig1852:0-2203
A1.2
DC.1
0
168
Contig1972:29-1001
A1.2
DC.1
7.11E-06
169
Contig2157:15-764
A1.2
DC.1
3.79E-08
170
Contig2380:0-1787
A1.2
DC.1
0.00925958
171
Contig3057:0-1927
A1.2
DC.1
1.84E-08
172
Contig5601:0-759
A1.2
DC.1
1.97E-05
173
Contig5821:0-949
A1.2
DC.1
3.71E-06
174
Contig9460:0-1091
A1.2
DC.1
6.54E-05
175
Contig9658:53-1094
A1.2
DC.1
7.28E-05
176
Contig10867:0-1043
A1.1
DC.1
0.000300206
177
Contig3512:0-616
A1.1
DC.1
0.0020524
178
Contig5184:30-251
A1.1
DC.1
0.000127465
179
Contig10271:0-621
A1.2
DC.1
0.000345329
180
Contig1514:18-187
A1.2
DC.1
0.00957196
181
Contig851:0-1951
A1.2
DC.1
0.000573248
182
Contig10081:0-821
A2.1
DC.1
5.97E-08
183
Contig161:0-979
A2.1
DC.1
5.85E-05
184
Contig1852:0-2203
A2.1
DC.1
0
185
Contig1972:29-1001
A2.1
DC.1
7.14E-06
186
Contig2157:15-764
A2.1
DC.1
4.11E-10
187
Contig3057:0-1927
A2.1
DC.1
1.37E-09
188
Contig5601:0-759
A2.1
DC.1
1.37E-06
189
Contig5624:0-2559
A2.1
DC.1
0.00684456
190
Contig5821:0-949
A2.1
DC.1
2.92E-05
191
Contig584:0-908
A2.1
DC.1
0.00560855
192
Contig7019:0-704
A2.1
DC.1
4.54E-05
193
Contig7251:0-1059
A2.1
DC.1
3.74E-05
194
Contig7708:0-1368
A2.1
DC.1
1.98E-05
195
Contig851:0-1951
A2.1
DC.1
0.000457521
196
Contig9460:0-1091
A2.1
DC.1
0.000428194
197
Contig10081:0-821
A2.2
DC.1
6.10E-08
198
Contig161:0-979
A2.2
DC.1
6.25E-05
199
Contig1852:0-2203
A2.2
DC.1
0
200
Contig1907:54-728
A2.2
DC.1
3.91E-06
201
Contig2139:0-1197
A2.2
DC.1
0.0329254
202
Contig2157:15-764
A2.2
DC.1
4.70E-10
203
Contig3057:0-1927
A2.2
DC.1
1.20E-09
204
Contig359:18-539
A2.2
DC.1
0.000271045
205
Contig4155:0-1303
A2.2
DC.1
0.00146942
206
Contig451:21-2271
A2.2
DC.1
2.48E-05
207
Contig462:0-927
A2.2
DC.1
6.10E-08
208
Contig7251:0-1059
A2.2
DC.1
1.64E-05
209
Contig7708:0-1368
A2.2
DC.1
2.81E-05
210
Contig851:0-1951
A2.2
DC.1
0.000567366
211
Contig2006:0-880
AC.1
DC.1
2.14E-05
212
Contig231:0-650
AC.1
DC.1
4.06E-06
213
Contig3946:2-446
AC.1
DC.1
4.75E-06
214
Contig418:2-2747
AC.1
DC.1
0.00570961
215
Contig4225:0-563
AC.1
DC.1
0.00025784
216
Contig4704:0-1313
AC.1
DC.1
1.03E-06
217
Contig489:21-1070
AC.1
DC.1
0.00109131
218
Contig7251:0-1059
AC.1
DC.1
3.85E-07
219
Contig7255:2-1153
AC.1
DC.1
0.00249426
220
Contig7356:0-819
AC.1
DC.1
1.54E-06
221
Contig8369:11-1513
AC.1
DC.1
0.000153811
222
Contig8590:0-1715
AC.1
DC.1
2.26E-05
223
Contig8879:0-594
AC.1
DC.1
5.61E-05
224
Contig8881:0-761
AC.1
DC.1
9.90E-07
225
Contig8890:0-844
AC.1
DC.1
1.46E-06
226
Contig1845:0-590
AC.2
DC.1
7.03E-05
227
Contig2006:0-880
AC.2
DC.1
5.08E-05
228
Contig231:0-650
AC.2
DC.1
9.69E-06
229
Contig2380:0-1787
AC.2
DC.1
0.00027316
230
Contig3946:2-446
AC.2
DC.1
3.72E-06
231
Contig4225:0-563
AC.2
DC.1
0.000764351
232
Contig4704:0-1313
AC.2
DC.1
1.22E-06
233
Contig7019:0-704
AC.2
DC.1
0.000754809
234
Contig7251:0-1059
AC.2
DC.1
9.90E-07
235
Contig7356:0-819
AC.2
DC.1
4.73E-06
236
Contig8590:0-1715
AC.2
DC.1
6.97E-05
237
Contig8879:0-594
AC.2
DC.1
0.000120113
238
Contig8881:0-761
AC.2
DC.1
1.75E-06
239
Contig8890:0-844
AC.2
DC.1
3.19E-06
240
Contig8369:11-1513
AC.1
DC.2
0.000128175
241
Contig2380:0-1787
D1.1
DC.2
0.00154775
242
Contig2380:0-1787
D1.1
DC.1
0.00253405
243
Contig2867:23-797
D1.1
DC.1
5.25E-08
244
Contig3395:0-424
D1.1
DC.1
5.29E-08
245
Contig3512:0-616
D1.1
DC.1
7.81E-07
246
Contig362:0-275
D1.1
DC.1
3.74E-05
247
Contig3946:2-446
D1.1
DC.1
1.42E-06
248
Contig6427:0-816
D1.1
DC.1
2.00E-05
249
Contig6578:0-492
D1.1
DC.1
5.05E-05
250
Contig7008:0-1433
D1.1
DC.1
0.000150039
251
Contig7015:0-567
D1.1
DC.1
0.000161943
252
Contig7150:0-1455
D1.1
DC.1
2.97E-06
253
Contig7546:0-614
D1.1
DC.1
2.39E-05
254
Contig7708:0-1368
D1.1
DC.1
0.000175604
255
Contig848:0-283
D1.1
DC.1
2.87E-05
256
Contig8568:0-1291
D1.1
DC.1
1.58E-06
257
Contig2380:0-1787
D1.2
DC.1
0.00214506
258
Contig2867:23-797
D1.2
DC.1
7.11E-08
259
Contig3395:0-424
D1.2
DC.1
5.28E-08
260
Contig3512:0-616
D1.2
DC.1
6.17E-07
261
Contig362:0-275
D1.2
DC.1
3.24E-05
262
Contig3946:2-446
D1.2
DC.1
1.38E-06
6.4.2: Contigs of A.annua and A.dubia producing more than 90% homology
This study of transcriptome of A. annua and A. dubia first time allowed
sequence of functional genes. On the basis of established fact of evolution from
common ancestor and sequence conservation, this new transcriptomic sequence was
matched with genes with known functions and sequences in nr/nt database to predict
their putative molecular functions. Additionally, structures of protein and different
kind of ribonucleic acids can also be predicted for these newly sequenced genes.
Evolutionarily close relatives of A. annua and A. dubia can be proposed through
phylogenetic tree construction. Percentage similarity gives the idea about how far the
two species have lived in genetic time period.
In order to validate the sequences, BLAST of contigs, obtained through
alignment of sequences of different transcriptomic sequences of these Artemisia
species, was performed. 16400 contigs were generated by alignment of different
samples of A. annua and A. dubia. Only 500 contigs were selected for BLAST on the
basis of significant value and in order to properly manage data for the study. Of these
500 contigs, 264 showed homology with genes of different organisms. Only those
contigs were selected for analysis and putative gene functions that showed more than
90% homology.
BLAST results of Contig3276:0-518 showed that gene WRKY of Malus x
domestica has percentage similarity of 94%. This gene is a transcription factor that
plays a key role in artemisinin synthesis pathway in Artemisia. Similarly, BLAST
results of contigs Contig5597:0-1737 showed that genes CBS, CBS732, CBS6284 of
Naumovozyma dairenensis, Zygosaccharomyces rouxii, and Tetrapisispora blattae
respectively, Contig10105:0-1419 with gene CCPU of Cynara cardunculus and
Contig3946:2-446 with gene TX077308 of Francisella sp. have percentage similarity
92-100%. These genes are related to cytochrome that plays an important role in
artemisinin biosynthesis pathway. Sequences of contigs which showed 90% or more
homology were used for BLAST and for other studies are given in appendix VI.
Table 6.2: Sequences producing significant Homology
S.No
.
Contigs
Source
Gene
Putative Gene Function
Accession
No.
Transcription factor
HM122720.1
E-Value
%age
Similarity
A1 AND A2
1
2
3
Contig3276:0518
Malus x domestica
Contig57:9-676
Lactuca sativa
Contig5987:0452
Solanum
lycopersicum
Solanum
lycopersicum
Lycopersicon
esculentum
WRKY17
psaH
Docking of the LHC I
antenna complex
LEFL1016AF02
rRNA binding
FC11AF10
134016F
NADH dehydrogenase
activity
RNA-directed DNA
polymerase activity
0.001
94%
AF162205.1
4.00E102
91%
AK321003.1
2.00E-13
93%
AK246443.1
2.00E-13
93%
BT014562.1
1.00E-10
92%
A1 AND AC
5
6
Contig100:43844
Solanum nigrum
Metallothioneinlike mRNA
Metal ion binding
FJ402840.1
4.00E-07
95%
Flaveria bidentis
pNDK-b
ATP-binding, Kinase
Activity
U10283.1
1.00E167
90%
NDK
ATP-binding, Kinase
Activity
U72142.1
8.00E164
90%
Contig1606:211047
Helianthus annuus
7
8
10
Contig161:0979
Contig167:6215
Contig2157:15764
Brassica oleracea
DQ267199.1
6.00E-64
90%
95%
Flaveria anomala
gdcsPA
Oxidoreductase
Z36879.1
Flaveria anomala
gdcsP
Oxidoreductase
Z99762.1
0.00E+0
0
95%
Flaveria anomala
gdcsPB gene
Oxidoreductase
Z54239.1
1.00E-64
95%
Vitis vinifera
ENTAV
Signal transducer activity
AM485693.1
8.00E-41
91%
Ajellomyces
capsulatus
HCAG_07571
Decarboxylating and
phosphate binding
XM_001537
212.1
1.00E-08
94%
Artemisia annua
CAZI5271
Structural constituent of
ribosome
EY083474.1
0.00E+0
0
95%
Artemisia annua
CAZI5271
Structural constituent of
ribosome
EY107471.1
7.00E-31
100%
Coffea arabica
CA00-XX-FB2022-A07-JE
Nucleotidyltransferase
GT702383.1
1.00E-04
97%
Koi herpesvirus
Contig5597:01737
CA-responsive protein
0.00E+0
0
Cyprinid
herpesvirus
11
BoCAR44
Naumovozyma
dairenensis
Zygosaccharomyce
s rouxii
FL ORF148
Membrane protein ORF148
GU815504.1
3.00E-07
97%
KHV-I
Serine-type endopeptidase
activity
DQ177346.1
3.00E-07
100%
CBS
Activator of cytochrome b
HE580273.1
1.00E-06
97%
CBS732
Activator of cytochrome b
CU928173.1
1.00E-06
97%
Tetrapisispora
blattae
Kuenenia
stuttgartiensis
CBS 6284
Activator of cytochrome b
HE806321.1
1.00E-05
97%
KUST_C
Repressor
CT573073.1
1.00E-05
97%
Pantoea ananatis
PA13
Catalytic activity
CP003085.1
2.00E-04
100%
Thielavia terrestris
NRRL 8126
Shikimate kinase activity
CP003011.1
2.00E-04
97%
Zygosaccharomyce
s rouxii
CBS732
MAP kinase activity
CU928175.1
2.00E-04
100%
Neurospora crassa
B11B23
Pyridoxal phosphate binding
AL669991.1
2.00E-04
100%
Trypanosoma
brucei gambiense
DAL972
Endonuclease activity
FN554971.1
6.00E-04
100%
Capsicum annuum
CaCM529L23
Vasodilation
GU048888.1
0.002
100%
C08HBa0210J04
Exonuclease
AP009282.1
0.002
100%
CgPTK-l
ATP binding
AB098199.1
4.00E-06
97%
DDB_G0280785
DNA binding
4.00E-06
97%
Puccinia graminis
ketoacyl-CoA
thiolase A
2.00E-04
100%
Solanum
lycopersicum
LEFL1039CB09
Acetyl-CoA CAcyltransferase activity
Receptor tyrosine-protein
kinase
XM_635935.
1
XM_003329
597.1
AK322583.1
6.00E-04
94%
Drosophila virilis
Dvir\GJ13854
Complete genome
6.00E-04
94%
Arabidopsis
thaliana
IKU1
Regulation of seed growth
XM_002048
196.1
NM_129075.
3
0.002
100%
Solanum
lycopersicum
Codonosiga
gracilis
Dictyostelium
discoideum
12
Contig2380:01787
EY104210.1
6.00E133
96%
Artemisia annua
CAZI3603
Structural constituent of
ribosome
Lotus japonicus
BP056955
DNA binding
GO022661.1
2.00E-08
97%
Solanum
tuberosum
POADE79
Trichodiene synthase activity
CK274023.1
5.00E-05
97%
Pinus pinaster
PPSW21
Oxidoreductase
BX000758.1
5.00E-05
95%
Honey bee
BH10057G22
RNA binding
DB744146.1
2.00E-04
100%
Cynara
cardunculus
CCPX3860
Serine-type endopeptidase
activity
GE606757.1
6.00E-04
94%
Artemisia annua
CAZI23588
Structural constituent of
ribosome
EY080578.1
0.00E+0
0
98%
91%
Lactuca virosa
CLVY6837
Calcium ion binding
DW168485.1
0.00E+0
0
Centaurea
solstitialis
CNSM11838
ATP Binding
EH765839.1
0.00E+0
0
92%
91%
Taraxacum
officinale
CTOY4902
DNA binding
DY829234.1
0.00E+0
0
Centaurea
maculosa
CNMM5610
DNA binding
EH732731.1
0.00E+0
0
91%
Helianthus exilis
CHEM3111
Transducer
EE643217.1
0.00E+0
90%
0
13
14
Contig249:21956
Contig253:01100
Contig3121:0262
15
3.00E-15
92%
Structural constituent of
ribosome
XM_003608
228.1
XM_001265
256.1
1.00E-13
93%
ATCC 42464
Hydrolase activity
CP003005.1
2.00E-16
95%
Pisum sativum
PUB2
Ubiquitin-protein ligase
activity
L81140.1
4.00E-14
92%
Ricinus communis
FBA, putative
mRNA
Hydro-lyase activity
XM_002528
547.1
1.00E-06
90%
Rattus norvegicus
Col18a1
Structural molecule activity
2.00E-06
Drosophila virilis
Dvir\GJ16689
Complete genome
NM_053489.
2
XM_002056
755.1
2.00E-06
97%
Zea mays clone
EL01N0361G07.
c
rRNA binding
BT017098.1
2.00E-06
97%
Drosophila
persimilis
Dper\GL14534
Zinc ion binding
8.00E-06
97%
Drosophila virilis
Dvir\GJ18864
Complete genome
1.00E-04
100%
Drosophila erecta
Dere\GG17580
Complete genome
1.00E-04
97%
Oryza sativa
Os06g0589800
ATP Binding
XM_002022
800.1
XM_002055
268.1
XM_001978
582.1
NM_001064
499.1
1.00E-04
95%
Mus musculus
RP23-365N23
RNA binding
AC159893.2
5.00E-08
95%
Medicago
truncatula
Neosartorya
fischeri
Myceliophthora
thermophila
MTR_4g091580
PTM
NFIA_020680
95%
Mouse DNA
RP23-123H11
RNA binding
CT030637.9
2.00E-07
95%
Rattus norvegicus
RNECO-134C12
DNA binding
AC245859.6
2.00E-06
97%
Candida
parapsilosis
Human
chromosome
Drosophila
melanogaster
CDC317
Oxidoreductase
HE605203.1
2.00E-06
97%
BAC R-909M7
Antibiotic
AL132709.5
2.00E-06
95%
BACR-32D05
Antibiotic
8.00E-06
100%
Mouse DNA
RP23-80C18
RNA binding
AC010212.1
0
AL590864.1
2
8.00E-06
97%
ATCC 42464
Hydrolase activity
CP003007.1
3.00E-05
100%
384-01M13F.t2F_J01
LjT02G13,
TM0489a
Sequence-specific DNA
binding
HQ437322.1
1.00E-04
95%
DNA binding
AP009827.1
1.00E-04
97%
Otocolobus manul
SRY
Activator, Repressor
DQ095178.1
1.00E-04
97%
Echinometra sp.
T-Ftm5_A1
Binding Function
AY261271.1
1.00E-04
100%
Artemisia annua
CAZI5271
Structural constituent of
ribosome
EY083474.1
0.00E+0
0
95%
Artemisia annua
CAZI5271
Structural constituent of
ribosome
EY107471.1
7.00E-31
100%
Myceliophthora
thermophila
Alexandrium
ostenfeldii
Lotus japonicus
A2 AND AC
16
Contig2157:15764
17
18
19
Contig8369:111513
Contig2380:01787
Contig5597:01737
Coffea arabica
CA00-XX-FB2022-A07-JE
Artemisia annua
CTOX5296.b1_
P04
Artemisia annua
Lactuca virosa
GT702383.1
1.00E-04
97%
Integral to membrane
EY115636.1
1.00E163
95%
CAZI23588
Structural constituent of
ribosome
EY080578.1
0.00E+0
0
98%
CLVY6837
Calcium ion binding
DW168485.1
0.00E+0
0
91%
92%
RNA binding
Centaurea
solstitialis
CNSM11838
ATP Binding
EH765839.1
0.00E+0
0
Taraxacum
officinale
CTOY4902
DNA binding
DY829234.1
0.00E+0
0
91%
91%
90%
Centaurea
maculosa
CNMM5610
DNA binding
EH732731.1
0.00E+0
0
Helianthus exilis
CHEM3111
Transducer
EE643217.1
0.00E+0
0
Cyprinid
herpesvirus
FL ORF148
Membrane protein ORF148
GU815504.1
Koi herpesvirus
KHV-I
ATP Binding
DQ177346.1
3.00E-07
100%
Naumovozyma
dairenensis
CBS
Translation regulator activity
HE580273.1
1.00E-06
97%
3.00E-07
97%
Zygosaccharomyce
s rouxii
Tetrapisispora
blattae
Kuenenia
stuttgartiensis
CBS732
Translation regulator activity
CU928173.1
1.00E-06
97%
CBS 6284
Translation regulator activity
HE806321.1
1.00E-05
97%
KUST_C
Repressor
CT573073.1
1.00E-05
97%
Pantoea ananatis
PA13
Catalytic activity
CP003085.1
2.00E-04
100%
Thielavia terrestris
NRRL 8126
Shikimate kinase activity
CP003011.1
2.00E-04
100%
Zygosaccharomyce
s rouxii
CBS732
Translation regulator activity
CU928175.1
2.00E-04
100%
Neurospora crassa
B11B23
Pyridoxal phosphate binding
AL669991.1
2.00E-04
100%
Trypanosoma
brucei gambiense
DAL972
Endonuclease activity
FN554971.1
6.00E-04
100%
Capsicum annuum
CaCM529L23
Vasodilation
GU048888.1
0.002
100%
C08HBa0210J04
Exonuclease
AP009282.1
0.002
100%
CgPTK-l
ATP binding
AB098199.1
4.00E-06
97%
DDB_G0280785
DNA binding
4.00E-06
97%
Puccinia graminis
ketoacyl-CoA
thiolase A
2.00E-04
100%
Solanum
lycopersicum
LEFL1039CB09
Acetyl-CoA CAcyltransferase activity
Receptor tyrosine-protein
kinase
XM_635935.
1
XM_003329
597.1
AK322583.1
6.00E-04
94%
Drosophila virilis
Dvir\GJ13854
XM_002048
196.1
6.00E-04
94%
Solanum
lycopersicum
Codonosiga
gracilis
Dictyostelium
discoideum
Complete genome
NM_129075.
3
0.002
100%
CAZI3603
Structural constituent of
ribosome
EY104210.1
6.00E133
96%
Lotus japonicus
BP056955
DNA binding
GO022661.1
Solanum
tuberosum
POADE79
Trichodiene synthase activity
CK274023.1
5.00E-05
97%
Pinus pinaster
PPSW21
Oxidoreductase
BX000758.1
5.00E-05
95%
Honey bee
BH10057G22
RNA binding
DB744146.1
2.00E-04
100%
Cynara
cardunculus
CCPX3860
GE606757.1
6.00E-04
94%
Artemisia annua
CAZI3763.CAZI
EY104502.1
0
99%
Cynara
cardunculus
CCPU963
Heme binding, cytochrome c
GE585313.1
2.00E-15
92%
Brassica oleracea
BoCAR44
CA-responsive protein
DQ267199.1
6.00E-64
90%
Drosophila yakuba
Dyak\GE24309
Complete proteome
XM_002097
700.1
4.00E-04
95%
Francisella sp.
TX077308
Complete proteome
CP002872.1
0.001
100%
KLTH0F07854g
Zinc ion binding
XM_002554
498.1
0.001
100%
CBS 6340
Translation regulator activity
CU928170.1
0.001
100%
Arabidopsis
thaliana
20
21
22
Contig10105:01419
Contig161:0979
Contig3946:2446
IKU1
Regulation of seed growth
Artemisia annua
Lachancea
thermotolerans
Lachancea
thermotolerans
Serine-type endopeptidase
activity
Structural constituent of
ribosome
2.00E-08
97%
XM_002138
246.1
XM_002054
269.1
XM_002023
391.1
0.001
100%
0.001
100%
0.001
100%
Chaperone
CP000937.1
0.001
92%
IP09721
Sequence-specific DNA
binding
BT029275.1
0.001
100%
Eriocheir sinensis
Esc29
Repressor
DQ450915.1
0.001
100%
Roridula dentata
CONN12998
DNA binding
JQ519390.1
3.00E-36
97%
Asclepias subulata
OKLA
JN665094.1
3.00E-36
97%
Daucus carota
BAC C237E06
FJ150367.1
3.00E-36
97%
Camellia sinensis
XUTDF2
DNA binding
EU730587.1
3.00E-36
97%
Solanum
lycopersicum
hba-17c15 map 1
Serine/threonine-protein
kinase
AC246154.3
5.00E-96
90%
Candida glabrata
CBS138
Translation regulator activity
CR380959.2
4.00E-09
92%
Clavispora
lusitaniae
ATCC 42720
Chaperone
XM_002619
926.1
8.00E-11
94%
Homo sapiens
PRKCE
ATP binding
EU332867.1
0.005
100%
Zebrafish
CH73-269F15
Endopeptidase inhibitor
activity
CU694370.9
0.005
100%
Drosophila
pseudoobscura
Dpse\GA24674
Complete proteome
Drosophila virilis
Dvir\GJ22874
Complete genome
Dper\GL20353
Zinc ion binding
ATCC 25017
Drosophila
persimilis
Francisella
philomiragia
Drosophila
melanogaster
23
24
25
Contig515:13967
Contig8590:01715
Contig3840:01556
NADH dehydrogenase
activity
Defense response to
bacterium
26
27
Contig4225:0563
Contig4679:38421
Homo sapiens
RP11-457N9
Structural constituent of
ribosome
AC017078.8
0.005
100%
Pan troglodytes
PTB-044H21
Nucleotide binding
AL954251.1
0.005
100%
Arabidopsis
thaliana
F19K19
ATP Binding
AC011808.4
5.00E-06
93%
Pig DNA sequence
CH242-209D19
Zinc ion binding
FP085370.17
0.003
100%
Mus musculus
EUCOMM
Integral to membrane
JN962323.1
0.011
100%
Pan troglodytes
CH251-324M23
Fatty acid transporter activity
AC197406.4
0.011
100%
Homo sapiens
MLLT10
Zinc ion binding
NG_027818.
1
0.011
100%
Macaca mulatta
CH250-530I2
Cholesterol 25-hydroxylase
activity
AC205183.3
0.011
100%
Mouse
RP24-145F3
RNA binding
0.011
100%
Zebrafish
DKEY-184E21
Oxidoreductase activity
CT030159.1
1
CT027699.1
4
0.011
100%
Macrohasseltia
macroterantha
trnL
RNA binding
AY756924.1
0.011
100%
Homo sapiens
CTD-2303L17
Carboxy-terminal domain
protein kinase
AC127034.2
0.011
100%
Arabidopsis
thaliana
BAC F19K19
Complete proteome
AC011808.4
5.00E-06
93%
Pig DNA sequence
CH242-209D19
Zinc ion binding
FP085370.17
0.003
100%
Mus musculus
EUCOMM
Integral to membrane
JN962323.1
0.011
100%
28
Contig5845:0796
Pan troglodytes
CH251-324M23
Fatty acid transporter activity
AC197406.4
0.011
100%
Homo sapiens
MLLT10
Zinc ion binding
NG_027818.
1
0.011
100%
Macaca mulatta
CH250-530I2
Cholesterol 25-hydroxylase
activity
AC205183.3
0.011
100%
Mouse
RP24-145F3
RNA binding
0.011
100%
Zebrafish
DKEY-184E21
Oxidoreductase activity
CT030159.1
1
CT027699.1
4
0.011
100%
Macrohasseltia
macroterantha
trnL
RNA binding
AY756924.1
0.011
100%
Homo sapiens
CTD-2303L17
Carboxy-terminal domain
protein kinase
AC127034.2
0.011
100%
Drosophila
persimilis
Dper\GL10834
Zinc ion binding
XM_002015
746.1
6.00E-04
100%
Oryza sativa
OSJNBa0023G1
1
ATP binding
AL845345.9
6.00E-04
100%
Artemisia annua
CAZI3763.CAZI
structural constituent of
ribosome
EY104502.1
0
99%
CCPU963
CA-responsive protein
GE585313.1
2.00E-15
92%
F19K19
ATP Binding
AC011808.4
5.00E-06
93%
CH242-209D19
Zinc ion binding
FP085370.17
0.003
100%
A1 AND D1
29
30
Contig10105:01419
Contig4225:0563
Cynara
cardunculus
Arabidopsis
thaliana
Pig DNA sequence
31
Contig4679:38421
Mus musculus
EUCOMM
Integral to membrane
JN962323.1
0.011
100%
Pan troglodytes
CH251-324M23
Fatty acid transporter activity
AC197406.4
0.011
100%
Homo sapiens
MLLT10
Zinc ion binding
NG_027818.
1
0.011
100%
Macaca mulatta
CH250-530I2
Cholesterol 25-hydroxylase
activity
AC205183.3
0.011
100%
Mouse
RP24-145F3
RNA binding
0.011
100%
Zebrafish
DKEY-184E21
Oxidoreductase activity
CT030159.1
1
CT027699.1
4
0.011
100%
Macrohasseltia
macroterantha
trnL
RNA binding
AY756924.1
0.011
100%
Homo sapiens
CTD-2303L17
Carboxy-terminal domain
protein kinase
AC127034.2
0.011
100%
Arabidopsis
thaliana
BAC F19K19
Complete proteome
AC011808.4
5.00E-06
93%
Pig DNA sequence
CH242-209D19
Zinc ion binding
FP085370.17
0.003
100%
Mus musculus
EUCOMM
Integral to membrane
JN962323.1
0.011
100%
Pan troglodytes
CH251-324M23
Fatty acid transporter activity
AC197406.4
0.011
100%
Homo sapiens
MLLT10
Zinc ion binding
NG_027818.
1
0.011
100%
Macaca mulatta
CH250-530I2
Cholesterol 25-hydroxylase
activity
AC205183.3
0.011
100%
Mouse
RP24-145F3
RNA binding
CT030159.1
1
0.011
100%
32
33
34
35
36
37
38
Contig296:0295
Contig5601:0759
Contig718:01984
Contig7540:01080
Contig8220:0844
Contig209:0247
Contig249:21956
Zebrafish
DKEY-184E21
Oxidoreductase activity
CT027699.1
4
0.011
100%
Macrohasseltia
macroterantha
trnL
RNA binding
AY756924.1
0.011
100%
Homo sapiens
CTD-2303L17
Carboxy-terminal domain
protein kinase
AC127034.2
0.011
100%
Drosophila yakuba
Dyak\GE12865
Complete proteome
XM_002090
280.1
3.00E-04
100%
Mus musculus
RP23-353C21
RNA binding
AC161815.4
0.001
100%
Venturia canescens
Vcan025
Transporter activity
DQ649259.1
0.003
100%
Che8
Mycobacterium phage
AY129330.1
0.003
100%
Gp96
Caviid herpesvirus 2
NM_214643.
2
0.007
100%
Mycobacteriopahg
e
Strongylocentrotus
purpuratus
Potassium channel regulator
activity
Sucrose: hydrogen symporter
activity
FJ119030.1
3.00E-10
94%
XM_002893
200.1
0.002
97%
R11198
ATP Binding
BT000684.1
0.002
97%
RP24-74L7
RNA binding
AC155164.5
0.004
100%
defensin BD2
Antibiotic
FR873267.1
3.00E-04
100%
MTR_4g091580
PTM
XM_003608
228.1
3.00E-15
92%
Pinus taeda
UMN_CL124
Arabidopsis lyrata
SUC2
Arabidopsis
thaliana
Mus musculus
Mytilus
galloprovincialis
Medicago
truncatula
NFIA_020680
Structural constituent of
ribosome
XM_001265
256.1
1.00E-13
93%
ATCC 42464
Hydrolase activity
CP003005.1
2.00E-16
95%
Pisum sativum
PUB2
Ubiquitin-protein ligase
activity
L81140.1
4.00E-14
92%
Drosophila
grimshawi
Dgri\GH22070
Complete proteome
3.00E-05
100%
Drosophila yakuba
Dyak\GE24309
Complete proteome
XM_001987
669.1
XM_002097
700.1
4.00E-04
95%
Francisella sp.
TX077308
Heme binding, cytochrome c
CP002872.1
0.001
100%
KLTH0F07854g
Translation regulator activity
XM_002554
498.1
0.001
100%
CBS 6340
Motor Protein
CU928170.1
0.001
100%
Dpse\GA24674
Complete genome
0.001
100%
Dvir\GJ22874
Zinc ion binding
XM_002138
246.1
XM_002054
269.1
0.001
100%
ATCC 25017
Chaperone
CP000937.1
0.001
92%
IP09721
Sequence-specific DNA
binding
BT029275.1
0.001
100%
Eriocheir sinensis
Esc29
Repressor
DQ450915.1
0.001
100%
Cyprinid
herpesvirus
FL ORF148
Membrane protein ORF148
GU815504.1
3.00E-07
97%
Neosartorya
fischeri
Myceliophthora
thermophila
39
Contig3946:2446
Lachancea
thermotolerans
Lachancea
thermotolerans
Drosophila
pseudoobscura
Drosophila virilis
Francisella
philomiragia
Drosophila
melanogaster
40
Contig5597:01737
Koi herpesvirus
Naumovozyma
dairenensis
Zygosaccharomyce
s rouxii
Tetrapisispora
blattae
Kuenenia
stuttgartiensis
Pantoea ananatis
KHV-I
ATP Binding
DQ177346.1
3.00E-07
100%
CBS
Translation regulator activity
HE580273.1
1.00E-06
97%
CBS732
Translation regulator activity
CU928173.1
1.00E-06
97%
CBS 6284
Translation regulator activity
HE806321.1
1.00E-05
97%
KUST_C
Repressor
CT573073.1
1.00E-05
97%
PA13
Catalytic activity
CP003085.1
2.00E-04
100%
Shikimate kinase activity
CP003011.1
2.00E-04
100%
Thielavia terrestris NRRL 8126
Zygosaccharomyce
s rouxii
CBS732
Translation regulator activity
CU928175.1
2.00E-04
100%
Neurospora crassa
B11B23
Pyridoxal phosphate binding
AL669991.1
2.00E-04
100%
Trypanosoma
brucei gambiense
DAL972
Endonuclease activity
FN554971.1
6.00E-04
100%
Capsicum annuum
CaCM529L23
Vasodilation
GU048888.1
0.002
100%
C08HBa0210J04
Exonuclease
AP009282.1
0.002
100%
CgPTK-l
ATP binding
AB098199.1
4.00E-06
97%
DDB_G0280785
DNA binding
4.00E-06
97%
ketoacyl-CoA
thiolase A
Acetyl-CoA CAcyltransferase activity
XM_635935.
1
XM_003329
597.1
2.00E-04
100%
Solanum
lycopersicum
Codonosiga
gracilis
Dictyostelium
discoideum
Puccinia graminis
Solanum
lycopersicum
LEFL1039CB09
Receptor tyrosine-protein
kinase
Drosophila virilis
Dvir\GJ13854
Complete genome
Arabidopsis
thaliana
IKU1
Regulation of seed growth
Artemisia annua
CAZI3603
Structural constituent of
ribosome
Lotus japonicus
BP056955
Solanum
tuberosum
AK322583.1
6.00E-04
94%
XM_002048
196.1
NM_129075.
3
6.00E-04
94%
0.002
100%
EY104210.1
6.00E133
96%
DNA binding
GO022661.1
2.00E-08
97%
POADE79
Trichodiene synthase activity
CK274023.1
5.00E-05
97%
Pinus pinaster
PPSW21
Oxidoreductase
BX000758.1
5.00E-05
95%
Honey bee
BH10057G22
RNA binding
DB744146.1
2.00E-04
100%
GE606757.1
6.00E-04
94%
GE606757.1
6.00E-04
94%
Cynara
cardunculus
Cynara
cardunculus
CCPX3860
CCPX3860
Serine-type endopeptidase
activity
Serine-type endopeptidase
activity
A2 AND D1
41
Contig10105:01419
Artemisia annua
CAZI3763.CAZI
Structural constituent of
ribosome
EY104502.1
0
99%
Cynara
cardunculus
CCPU963
Heme binding, cytochrome c
GE585313.1
2.00E-15
92%
42
Contig296:0295
Drosophila yakuba
Dyak\GE12865
Complete proteome
XM_002090
280.1
3.00E-04
100%
Mus musculus
RP23-353C21
RNA binding
AC161815.4
0.001
100%
Venturia canescens
Vcan025
Transporter activity
DQ649259.1
0.003
100%
Che8
Mycobacterium phage
AY129330.1
0.003
100%
Dgri\GH22070
Complete proteome
3.00E-05
100%
Drosophila yakuba
Dyak\GE24309
Complete proteome
XM_001987
669.1
XM_002097
700.1
4.00E-04
95%
Francisella sp.
TX077308
Heme binding, cytochrome c
CP002872.1
0.001
100%
KLTH0F07854g
Translation regulator activity
XM_002554
498.1
0.001
100%
CBS 6340
Motor Protein
CU928170.1
0.001
100%
Dpse\GA24674
Complete genome
0.001
100%
Dvir\GJ22874
Zinc ion binding
XM_002138
246.1
XM_002054
269.1
0.001
100%
ATCC 25017
Chaperone
CP000937.1
0.001
92%
IP09721
Sequence-specific DNA
binding
BT029275.1
0.001
100%
Eriocheir sinensis
Esc29
Repressor
DQ450915.1
0.001
100%
Medicago
truncatula
mte1-58c24
Transcription factor
CR955005.2
4.00E-12
93%
Mycobacteriopahg
e
Drosophila
grimshawi
43
Contig3946:2446
Lachancea
thermotolerans
Lachancea
thermotolerans
Drosophila
pseudoobscura
Drosophila virilis
Francisella
philomiragia
Drosophila
melanogaster
44
Contig4155:01303
45
46
Contig451:212271
Contig5601:0759
Homo sapiens
RP11-2B15
Ribonucleoprotein
AC009446.1
1
1.00E-05
100%
Mouse DNA
RP23-278I21
Ribonucleoprotein
AL596215.7
1.00E-05
100%
Mus musculus
KOMP
Nodulation protein
JN959018.1
4.00E-05
100%
Homo sapiens
TRAPPC9
Activator of NF-kappa-B
NG_016478.
1
4.00E-05
97%
Mus musculus
BAC RP23118E20
Component of MHC I
AC153894.8
4.00E-05
100%
Homo sapiens
CCBE1
Calcium ion binding
2.00E-04
100%
Pig DNA
CH242-140A18
Hydrolase
NG_016990.
1
CU062440.4
1
2.00E-04
100%
Pan troglodytes
CH251-272H6
Fatty acid transporter activity
AC193771.2
2.00E-04
100%
Gallus gallus
CH261-88N9
ATP binding
AC186838.4
2.00E-04
100%
Human DNA
MDN1, RPL22,
CASP8AP2
Ribonucleoprotein
2.00E-04
100%
Homo sapiens
ZMIZ1
Transcription factor
AL353692.1
4
NG_028289.
1
6.00E-04
100%
Macaca mulatta
CH250-188A12
Receptor
AC214204.5
6.00E-04
95%
Rhesus Macaque
CH250-352A6
Receptor
AC189964.2
6.00E-04
97%
Lotus japonicus
LjT09E08,
TM0265
DNA binding
AP006391.1
6.00E-04
100%
Strongylocentrotus
purpuratus
Gp96
Caviid herpesvirus 2
NM_214643.
2
0.007
100%
47
48
49
Contig7219:0736
Contig5987:0452
Contig5597:01737
Lotus japonicus
Solanum
lycopersicum
Solanum
lycopersicum
Lycopersicon
esculentum
Plasmodium
knowlesi
Cyprinid
herpesvirus
LjFL2-006BC04
DNA binding
AK337989.1
2.00E-10
94%
LEFL1016AF02
rRNA binding
AK321003.1
2.00E-13
93%
AK246443.1
2.00E-13
93%
BT014562.1
1.00E-10
92%
FC11AF10
134016F
NADH dehydrogenase
activity
RNA-directed DNA
polymerase activity
complete
genome
Complete genome
AM910986.1
2.00E-08
94%
FL ORF148
Membrane protein
GU815504.1
3.00E-07
97%
Geobacter sp
M18, complete
genome
DNA damage-binding
protein
CP002479.1
3.00E-07
100%
Koi herpesvirus
KHV-I
DNA polymerase
DQ177346.1
3.00E-07
100%
CBS
Trimethyllysine dioxygenase
HE580273.1
1.00E-06
97%
CU928173.1
1.00E-06
97%
CT573073.1
1.00E-05
97%
Naumovozyma
dairenensis
Zygosaccharomyce
s rouxii
Kuenenia
stuttgartiensis
CBS732
KUST_C
Mitogen-activated protein
kinase
Fragile X mental retardation
protein
Pantoea ananatis
PA13
DNA repair protein
CP003085.1
2.00E-04
100%
Thielavia terrestris
NRRL 8126
Transferase activity
CP003011.1
2.00E-04
100%
Zygosaccharomyce
s rouxii
CBS732
Mitogen-activated protein
kinase
CU928175.1
2.00E-04
100%
Neurospora crassa
B11B23
DNA binding
AL669991.1
2.00E-04
100%
Trypanosoma
brucei gambiense
DAL972
Nuclease activity
FN554971.1
6.00E-04
100%
Capsicum annuum
CaCM529L23
Transfer of acetyl-CoA
GU048888.1
0.002
100%
C08HBa0210J04
Carboxypeptidase
AP009282.1
0.002
100%
CgPTK-l
Tyrosine-protein kinase
AB098199.1
4.00E-06
97%
DDB_G0280785
DNA damage-binding
4.00E-06
97%
Puccinia graminis
ketoacyl-CoA
thiolase A
Acyltransferase
XM_635935.
1
XM_003329
597.1
2.00E-04
100%
Solanum
lycopersicum
LEFL1039CB09
Receptor tyrosine-protein
kinase
AK322583.1
6.00E-04
94%
Drosophila virilis
Dvir\GJ13854
Complete genome
6.00E-04
94%
Arabidopsis
thaliana
IKU1
Transciptional level
XM_002048
196.1
NM_129075.
3
0.002
100%
Artemisia annua
CAZI3603
Structural constituent of
ribosome
EY104210.1
6.00E133
96%
Lotus japonicus
BP056955
Catalytic activity
GO022661.1
2.00E-08
97%
EST004950
Telomerase complex
DW492938.1
5.00E-05
97%
POADE79
Trichodiene synthase activity
CK274023.1
5.00E-05
97%
PPSW21
Endonuclease activity
BX000758.1
5.00E-05
95%
Solanum
lycopersicum
Codonosiga
gracilis
Dictyostelium
discoideum
Gossypium
hirsutum
Solanum
tuberosum
Pinus pinaster
50
Honey bee
BH10057G22
RNA binding
DB744146.1
2.00E-04
100%
Cynara
cardunculus
CCPX3860
Target cell lysis
GE606757.1
6.00E-04
94%
Complete genome
EU549769.1
0
Complete genome
DQ383816.1
0
99%
Complete genome
DQ383815.1
0
99%
Complete genome
HQ234669.1
0
98%
ATPase
HQ664599.1
0
98%
Complete genome
DQ898156.1
0
98%
Complete genome
GU456628.1
0
97%
HM100310.1
0
98%
HM100311.1
0
98%
EU016780.1
0
98%
AB240139.1
0
98%
AC AND D1
Guizotia
abyssinica
Lactuca sativa
Helianthus annuus
Jacobaea vulgaris
Aucuba japonica
51
Contig10461:0582
Daucus carota
Anthriscus
cerefolium
Davidia
involucrata
Franklinia
alatamaha
Anethum
graveolens
Nicotiana
tomentosiformis
Complete
genome
Complete
genome
Complete
genome
Complete
genome
rpl23, trnI-CAU,
ycf2
Complete
genome
Complete
genome
ndhB
ndhB
ndhB
Complete
genome
Shuttles electrons from
NAD(P)H
Shuttles electrons from
NAD(P)H
Shuttles electrons from
NAD(P)H
Complete genome
99%
Datura
stramonium
Solanum
tuberosum
Solanum
bulbocastanum
Buxus microphylla
Coffea arabica
Olea europaea
Pachysandra
terminalis
Fosterella
caulescens
Japonolirion
osense
Doryanthes
palmeri
Complete
genome
Complete
genome
Complete
genome
Complete
genome
Complete
genome
Complete
genome
rps12, ndhB
ndhB
ndhB
ndhB
Pitcairnia feliciana
ndhB
Flagellaria indica
ndhB
Molineria
capitulata
ndhB
Musa acuminata
ndhB
Complete genome
JN654342.1
0
98%
Complete genome
JF772170.2
0
98%
Complete genome
DQ347958.1
0
98%
Complete genome
EF380351.1
0
98%
Complete genome
EF044213.1
0
98%
Complete genome
GU228899.2
0
97%
Ribonucleoprotein
AY237141.1
0
98%
Shuttles electrons from
NAD(P)H
Shuttles electrons from
NAD(P)H
Shuttles electrons from
NAD(P)H
Shuttles electrons from
NAD(P)H
Shuttles electrons from
NAD(P)H
Shuttles electrons from
NAD(P)H
Shuttles electrons from
NAD(P)H
HQ180947.1
0
97%
JQ069014.1
0
97%
JQ276504.1
0
97%
HQ180964.1
0
97%
HQ180946.1
0
97%
HQ180941.1
0
97%
EU017042.1
0
97%
52
Contig2126:01124
Shuttles electrons from
NAD(P)H
Shuttles electrons from
NAD(P)H
EU016718.1
0
97%
HQ180959.1
0
97%
Aminoacyl-tRNA synthetase
JN710464.1
0
97%
Os02g0594100
Transcription factor
NM_001053
839.1
2.00E-07
97%
Gossypium
hirsutum
MONCS2109
NF-kappa-B activation
GQ395086.1
6.00E-07
95%
Mus musculus
RP24-371P8
Ribonucleoprotein
AC111014.6
2.00E-06
95%
Prunus persica
KNOPE2
GU144519.1
8.00E-06
95%
Amborella
trichopoda
AT_SBa0088I11
AC243606.1
8.00E-06
95%
Zebrafish
DKEY-12H9
ATP binding
8.00E-06
97%
Mouse DNA
RP23-219C18
Ribonucleoprotein
AL954831.1
7
AL672141.1
2
8.00E-06
95%
CH252-230M14
Cholesterol biosynthetic
enzymes
AC239272.5
3.00E-05
97%
mth2-124i6
Transcription factor
AC157982.2
7
3.00E-05
97%
Y46E12BM
Binding
FO081797.1
3.00E-05
97%
bRB-333H21
Endonuclease
FO117624.1
3
1.00E-04
93%
Acorus americanus
ndhB
Neoastelia
spectabilis
ndhB
Asclepias macrotis
LSUS
Oryza sativa
Chlorocebus
aethiops
Medicago
truncatula
Caenorhabditis
elegans
Rat DNA
Sequence-specific DNA
binding
Cation transmembrane
transporter activity
53
54
55
Contig362:0275
Contig418:22747
Contig4225:0563
Rattus norvegicus
RNECO-252C13
TRANSCRIPTION
FACTOR
AC245774.3
1.00E-04
97%
Talaromyces
stipitatus
UbiD/Ubi4
Covalent Attachment
XM_002341
825.1
4.00E-08
100%
Aspergillus niger
An04c0190
Thiosulfate sulfurtransferase
activity
AM270080.1
4.00E-08
100%
Arabidopsis
thaliana
UBQ4
Covalent Attachment
NM_122069.
3
5.00E-07
97%
Glycine max
Subi-1
Covalent Attachment
D16248.1
8.00E-05
94%
mth2-72p17
Transcription factor
CT573507.2
8.00E-04
94%
F19K19
Transferase
AC011808.4
5.00E-06
93%
Pig DNA sequence
CH242-209D19
Nucleic acid binding
FP085370.17
0.003
100%
Mus musculus
EUCOMM
MHC class II antigen
JN962323.1
0.011
100%
Pan troglodytes
CH251-324M23
MHC II Membrane
AC197406.4
0.011
100%
Homo sapiens
MLLT10
Zinc ion binding
NG_027818.
1
0.011
100%
Macaca mulatta
CH250-530I2
Receptor
AC205183.3
0.011
100%
Vitis vinifera
VV78X115886.6
Isomerase
AM489072.2
0.011
100%
Mouse
RP24-145F3
Ribonucleoprotein
0.011
100%
Zebrafish
DKEY-184E21
Membrane component
CT030159.1
1
CT027699.1
4
0.011
100%
Medicago
truncatula
Arabidopsis
thaliana
56
Contig2380:01787
Macrohasseltia
macroterantha
trnL
RNA binding
AY756924.1
0.011
100%
Homo sapiens
CTD-2303L17
Carboxy-terminal domain
protein kinase
AC127034.2
0.011
100%
98%
Artemisia annua
CAZI23588
Beta-lactam antibiotic
EY080578.1
0.00E+0
0
Lactuca virosa
CLVY6837
ATP binding
DW168485.1
0.00E+0
0
91%
Centaurea
solstitialis
CNSM11838
Chaperone
EH765839.1
0.00E+0
0
92%
91%
Taraxacum
officinale
CTOY4902
RNA-binding
DY829234.1
0.00E+0
0
Centaurea
maculosa
CNMM5610
Shaping and sizing of PSM)
EH732731.1
0.00E+0
0
91%
Helianthus exilis
CHEM3111
Receptor for the attracting Laspartate
EE643217.1
0.00E+0
0
90%
Silene diclinis
X3
Rigid and resistant hair shaft
EU521745.1
5.00E-49
90%
Drosophila
willistoni
Dwil\GK14864
Component of nucleosome
XM_002065
045.1
4.00E-05
95%
Eimeria tenella
AMA1
Activator protein
JN032081.1
0.007
100%
A1 AND DC1
57
Contig1852:02203
58
59
Contig1852:02203
Contig2157:15764
60
Leptosphaeria
maculans
Solanum
lycopersicum
Medicago
truncatula
JN3 lm
Endonuclease
FP929126.1
0.007
100%
LEFL1007BC07
Wnt signaling pathway
AK320272.1
0.007
100%
MTR_4g113140
DNA binding
XM_003609
160.1
2.00E-06
97%
95%
Artemisia annua
CAZI5271
Ribosomal protein
EY083474.1
0.00E+0
0
Artemisia annua
CAZI5271
Ribosomal protein
EY107471.1
7.00E-31
100%
Coffea arabica
CA00-XX-FB2022-A07-JE
Nucleotidyltransferase
GT702383.1
1.00E-04
97%
Beta-lactam antibiotic
EY080578.1
0.00E+0
0
98%
91%
Artemisia annua
CAZI23588
61
Lactuca virosa
CLVY6837
ATP binding
DW168485.1
0.00E+0
0
62
Centaurea
solstitialis
CNSM11838
Chaperone
EH765839.1
0.00E+0
0
92%
63
Taraxacum
officinale
CTOY4902
RNA-binding
DY829234.1
0.00E+0
0
91%
64
Centaurea
maculosa
CNMM5610
shaping and sizing of PSM)
EH732731.1
0.00E+0
91%
Contig2380:01787
0
65
66
Contig5601:0759
Helianthus exilis
CHEM3111
Receptor for the attracting Laspartate
Strongylocentrotus
purpuratus
Gp96
Caviid herpesvirus 2
Zebrafish
67
Contig7708:01368
68
Contig1852:02203
69
Contig5184:30251
70
Contig1514:18187
CH73-166J22
EE643217.1
Endopeptidase inhibitor
activity
NM_214643.
2
CU856173.1
9
0.00E+0
0
90%
0.007
100%
2.00E-17
92%
Pongo abelii
CH276-98H12
Serine esterase
AC187773.2
2.00E-17
100%
Zebrafish
SLC8A2
Calcium: sodium antiporter
activity
BX890602.8
8.00E-17
93%
Rattus norvegicus
BN/SsNHsd/MC
W
Electron carrier activity
AC133262.3
3.00E-16
92%
Mus musculus
RP24-323H7
Nucleic acid binding
AC102594.1
5
1.00E-15
91%
Macaca mulatta
DRB
Immune system
AM910410.1
1.00E-15
90%
Atelerix albiventris
LBNL4-89B6
Positive regulator of the
HHSP
AC144402.2
4.00E-15
91%
Silene diclinis
X3
Keratin-associated protein
EU521745.1
5.00E-49
90%
Mouse DNA
RP23-407I21
Isomerase, topoisomerase
9.00E-05
100%
Canis familiaris
XX-297J22
Probable collagen protein
0.001
97%
Giardia lamblia
GL50803_16310
Ribonucleoprotein
AL603706.1
3
AC188148.1
6
XM_001703
998.1
9.00E-09
91%
71
Contig851:01951
Senecio vernalis
eifsv1
Protein biosynthesis
AJ238624.1
3.00E173
Brassica oleracea
BoCAR44
Putative uncharacterized
protein
DQ267199.1
6.00E-64
90%
Silene diclinis
X3
Keratin-associated protein
EU521745.1
5.00E-49
90%
Artemisia annua
CAZI5271
Organization of the
cytomatrix
EY083474.1
0.00E+0
0
95%
Coffea arabica
CA00-XX-FB2022-A07-JE
RNA binding
GT702383.1
1.00E-04
97%
Gp96
Caviid herpesvirus 2
0.007
100%
Dper\GL10834
Zinc ion binding
NM_214643.
2
XM_002015
746.1
6.00E-04
100%
Oryza sativa
OSJNBa0023G1
1
zinc ion binding
AL845345.9
6.00E-04
100%
Zebrafish
CH73-166J22
Nucleic acid binding
CU856173.1
9
2.00E-17
92%
Pongo abelii
CH276-98H12
Serine esterase
AC187773.2
2.00E-17
100%
Zebrafish
SLC8A2
Calcium: sodium antiporter
activity
BX890602.8
8.00E-17
93%
Rattus norvegicus
BN/SsNHsd/MC
W
Electron carrier activity
AC133262.3
3.00E-16
92%
90%
A2 AND DC1
72
73
74
Contig161:0979
Contig1852:02203
Contig2157:15764
75
76
77
78
79
Contig5601:0759
Contig584:0908
Contig7708:01368
Strongylocentrotus
purpuratus
Drosophila
persimilis
Mus musculus
RP24-323H7
Zinc ion binding
AC102594.1
5
1.00E-15
91%
Macaca mulatta
DRB
MHC II BINDING
AM910410.1
1.00E-15
90%
Atelerix albiventris
LBNL4-89B6
Positive regulator of HHP
AC144402.2
4.00E-15
91%
80
Contig851:01951
Senecio vernalis
eifsv1
protein biosynthesis
AJ238624.1
3.00E173
90%
81
Contig4155:01303
Medicago
truncatula
mte1-58c24
Acyl-CoA thioesterases
CR955005.2
4.00E-12
93%
Homo sapiens
RP11-2B15
Ribonucleoprotein
AC009446.1
1
1.00E-05
100%
Mouse DNA
RP23-278I21
Ribonucleoprotein
AL596215.7
1.00E-05
100%
Mus musculus
KOMP
Nodulation protein
JN959018.1
4.00E-05
100%
Homo sapiens
TRAPPC9
Activator of NF-kappa-B
NG_016478.
1
4.00E-05
97%
Mus musculus
BAC RP23118E20
COMPONENT OF MHC I
AC153894.8
4.00E-05
100%
Homo sapiens
CCBE1
Calcium ion binding
2.00E-04
100%
Pig DNA
CH242-140A18
Hydrolase
NG_016990.
1
CU062440.4
1
2.00E-04
100%
Pan troglodytes
CH251-272H6
MHC II Membrane
AC193771.2
2.00E-04
100%
Gallus gallus
CH261-88N9
ATP binding
AC186838.4
2.00E-04
100%
82
Contig451:212271
83
84
Human DNA
MDN1, RPL22,
CASP8AP2
Ribonucleoprotein
2.00E-04
100%
TRANSCRIPTION
FACTOR
AL353692.1
4
NG_028289.
1
Homo sapiens
ZMIZ1
6.00E-04
100%
Macaca mulatta
CH250-188A12
Receptor
AC214204.5
6.00E-04
95%
Rhesus Macaque
CH250-352A6
Receptor
AC189964.2
6.00E-04
97%
Lotus japonicus
LjT09E08,
TM0265
DNA binding
AP006391.1
6.00E-04
100%
Drosophila
grimshawi
Dgri\GH22070
Complete proteome
3.00E-05
100%
Drosophila yakuba
Dyak\GE24309
Complete proteome
XM_001987
669.1
XM_002097
700.1
4.00E-04
95%
Francisella sp.
TX077308
Heme binding, cytochrome c
CP002872.1
0.001
100%
KLTH0F07854g
Translation regulator activity
XM_002554
498.1
0.001
100%
CBS 6340
Motor Protein
CU928170.1
0.001
100%
Dpse\GA24674
Complete genome
0.001
100%
Drosophila virilis
Dvir\GJ22874
Zinc ion binding
XM_002138
246.1
XM_002054
269.1
0.001
100%
Francisella
philomiragia
ATCC 25017
Chaperone
CP000937.1
0.001
92%
Drosophila
IP09721
Sequence-specific DNA
BT029275.1
0.001
100%
AC AND DC
Contig3946:2446
Lachancea
thermotolerans
Lachancea
thermotolerans
Drosophila
pseudoobscura
binding
melanogaster
Eriocheir sinensis
Esc29
Repressor
DQ450915.1
0.001
100%
85
Contig418:22747
Medicago
truncatula
mth2-72p17
Involved in biological aging
CT573507.2
8.00E-04
94%
86
Contig8369:111513
Artemisia annua
CTOX5296.b1_
P04
Thymocyte marker
EY115636.1
1.00E163
95%
87
Contig8590:01715
Solanum
lycopersicum
hba-17c15 map 1
Involved in oxygen transport
AC246154.3
5.00E-96
90%
Artemisia annua
CAZI22903
Beta-lactam antibiotic
EY096790.1
0
99%
88
Contig8879:0594
Chrysanthemum x
morifolium
LMS
Ribonucleoprotein
DK939321.1
2.00E-95
98%
Mouse DNA
Rps2
Disease resistance (R)
protein
AL772210.8
6.00E-06
97%
Rat DNA
bRB-233C6
Endonuclease
FO181541.1
1
7.00E-05
100%
Mizuhopecten
yessoensis
HLJX-141
Chaperone
FJ262385.1
7.00E-05
100%
Zebrafish
DKEY-153P21
Oxidoreductase activity
CU633478.1
0
7.00E-05
100%
Rattus norvegicus
SHR-Akr
Endopeptidase inhibitor
activity
AC242886.6
3.00E-04
97%
Pan troglodytes
CH251-381H2
Fatty acid transporter activity
AC192852.3
3.00E-04
100%
Mus musculus
RP24-127A22
Ribonucleoprotein
AC123833.3
3.00E-04
100%
Oryza sativa
OSJNBa0033D2
4
ATP binding
AP005439.3
3.00E-04
100%
89
Contig8881:0761
Tetrapisispora
phaffii
Gossypium
herbaceum
CBS 4417
Motor Protein
HE612859.1
0.001
100%
NBRI_M189
GTPase activity
JF495680.1
0.001
100%
Homo sapiens
FXYD6
Ion channel activity
NG_013071.
1
0.001
100%
Villosa fabalis
VfaB244
Hydrolase activity
GQ258763.1
0.001
100%
Candida
dubliniensis
CD36_06900
Protein-degradation mediator
XM_002417
302.1
0.001
100%
Felis catus
FCAB-91H12
Carbonate dehydratase
activity
AC234574.1
0.001
100%
Dictyostelium
discoideum
DDB_G0282573
DNA binding
XM_635042.
1
0.001
100%
Pan troglodytes
RP43-148A6
Fatty acid transporter activity
AC146001.2
0.001
100%
Zebrafish
CH211-218I18
Nucleic acid binding
AF324786.1
0.001
100%
Homo sapiens
RP11-728F11
Ribonucleoprotein
AP000757.4
0.001
100%
Weeksella virosa
DSM 16922
Nucleotidyltransferase
CP002455.1
0.003
100%
Pongo abelii
CH276-480A19
DNA topoisomerase activity
AC206881.3
0.003
100%
Thais clavigera
Tcl128
ATP synthase
FJ550296.1
Mus musculus
RP24-267D17
Ribonucleoprotein
AC123055.3
1.00E-05
100%
Homo sapiens
RP11-513N24
Ribonucleoprotein
AC022164.8
1.00E-05
100%
1.00E-05
90
Contig8890:0844
100%
91
92
Contig2380:01787
Contig4225:0563
Homo sapiens
PRKCB
Transferase, ATP Binding
NG_029003.
1
1.00E-05
100%
Gossypium
hirsutum
NBRI_Gh_J014
GTPase activity
HQ524507.1
0.002
97%
98%
Artemisia annua
CAZI23588
Beta-lactam antibiotic
EY080578.1
0.00E+0
0
Lactuca virosa
CLVY6837
ATP binding
DW168485.1
0.00E+0
0
91%
Centaurea
solstitialis
CNSM11838
Chaperone
EH765839.1
0.00E+0
0
92%
91%
Taraxacum
officinale
CTOY4902
RNA-binding
DY829234.1
0.00E+0
0
Centaurea
maculosa
CNMM5610
shaping and sizing of PSM)
EH732731.1
0.00E+0
0
91%
Helianthus exilis
CHEM3111
Receptor for the attracting Laspartate
EE643217.1
0.00E+0
0
90%
Arabidopsis
thaliana
F19K19
ATP Binding
AC011808.4
5.00E-06
93%
Pig DNA sequence
CH242-209D19
Zinc ion binding
FP085370.17
0.003
100%
Mus musculus
EUCOMM
Integral to membrane
JN962323.1
0.011
100%
Pan troglodytes
CH251-324M23
Fatty acid transporter activity
AC197406.4
0.011
100%
Homo sapiens
MLLT10
Zinc ion binding
NG_027818.
1
0.011
100%
Macaca mulatta
CH250-530I2
Cholesterol 25-hydroxylase
activity
AC205183.3
0.011
100%
Mouse
RP24-145F3
RNA binding
0.011
100%
Zebrafish
DKEY-184E21
Oxidoreductase activity
CT030159.1
1
CT027699.1
4
0.011
100%
Macrohasseltia
macroterantha
trnL
RNA binding
AY756924.1
0.011
100%
Homo sapiens
CTD-2303L17
Carboxy-terminal domain
protein kinase
AC127034.2
0.011
100%
Artemisia annua
CAZI23588
Beta-lactam antibiotic
EY080578.1
0.00E+0
0
98%
91%
D1 AND DC
93
Contig2380:01787
Lactuca virosa
CLVY6837
ATP binding
DW168485.1
0.00E+0
0
Centaurea
solstitialis
CNSM11838
Chaperone
EH765839.1
0.00E+0
0
92%
91%
91%
Taraxacum
officinale
CTOY4902
RNA-binding
DY829234.1
0.00E+0
0
Centaurea
maculosa
CNMM5610
shaping and sizing of PSM)
EH732731.1
0.00E+0
0
94
Contig362:0275
Helianthus exilis
CHEM3111
Receptor for the attracting Laspartate
EE643217.1
0.00E+0
0
90%
Talaromyces
stipitatus
UbiD/Ubi4
Covalent Attachment
XM_002341
825.1
4.00E-08
100%
Aspergillus niger
An04c0190
Transferase
AM270080.1
4.00E-08
100%
Arabidopsis
thaliana
UBQ4
Covalent Attachment
NM_122069.
3
5.00E-07
97%
Glycine max
Subi-1
Covalent Attachment
D16248.1
8.00E-05
94%
XM_001987
669.1
XM_002097
700.1
3.00E-05
100%
4.00E-04
95%
Drosophila
grimshawi
95
Contig3946:2446
Dgri\GH22070
Complete proteome
Drosophila yakuba
Dyak\GE24309
Complete proteome
Francisella sp.
TX077308
Heme binding, cytochrome c
CP002872.1
0.001
100%
KLTH0F07854g
Translation regulator activity
XM_002554
498.1
0.001
100%
CBS 6340
Motor Protein
CU928170.1
0.001
100%
Dpse\GA24674
Complete genome
0.001
100%
Drosophila virilis
Dvir\GJ22874
Zinc ion binding
XM_002138
246.1
XM_002054
269.1
0.001
100%
Francisella
philomiragia
ATCC 25017
Chaperone
CP000937.1
0.001
92%
Lachancea
thermotolerans
Lachancea
thermotolerans
Drosophila
pseudoobscura
96
97
Contig7708:01368
Contig848:0283
Drosophila
melanogaster
IP09721
Sequence-specific DNA
binding
BT029275.1
0.001
100%
Eriocheir sinensis
Esc29
Repressor
DQ450915.1
0.001
100%
Zebrafish
CH73-166J22
Nucleic acid binding
CU856173.1
9
2.00E-17
92%
Pongo abelii
CH276-98H12
Serine esterase
AC187773.2
2.00E-17
100%
Zebrafish
SLC8A2
Calcium: sodium antiporter
activity
BX890602.8
8.00E-17
93%
Rattus norvegicus
BN/SsNHsd/MC
W
Electron carrier activity
AC133262.3
3.00E-16
92%
Mus musculus
RP24-323H7
Zinc ion binding
AC102594.1
5
1.00E-15
91%
Macaca mulatta
DRB
MHC II BINDING
AM910410.1
1.00E-15
90%
Atelerix albiventris
LBNL4-89B6
Positive regulator of HHP
AC144402.2
4.00E-15
91%
Nicotiana tabacum
H-N_BC2M41322
DNA binding
DQ460104.1
4.00E-14
91%
Solanum
lycopersicum
C02HBa0091J18
Transferase activity
AC215381.1
9.00E-11
90%
Likewise results of BLAST of different contigs also showed many enzymes that
have established role in A.annua and A. dubia and were common to various other
species. Since the time of divergence of two species, a gene might have changed its
function it played in the ancestor, however, sequence still has certain homology with
its evolutionary related species.
BLAST of these transcriptomic sequences generated E-value that gives an idea of
relatedness of sequences. If an E value is below 0.01, then the match is significant.
Nonetheless, if the E value is higher, the match might still be significant especially for
a short sequence. In that case, results have to be evaluated in the context of
experiment.
This computational and predictive analysis of features, common amongst
different organisms, gives a reliable and significant amount of data. These results can
have many implications and can be extrapolated to understand different unknown
aspects for a newly sequenced genome.
There are many organisms that show 100% sequence conservation with
contigs generated through alignment of different samples of A. annua and A.dubia.
The genes of these organisms are well characterized and can be helpful in predicting
the molecular functions of genes of these Artemisia species.
6.4.4: Contigs of A.annua and A.dubia producing 80-85% homology
BLAST of Various contigs which were obtained by aligning different
transcriptomic sequences of A.annua and A.dubia, was performed and sequence
homology presented interesting results. These contigs showed homology with
different genome sequences ranging from 60% to 90% but only those Contigs were
analyzed that showed 90% homology or more than it (Table 6.2). However there are
many contigs which showed 80% and more homology produced valuable results.
Contigs showing 80% homology can be seen in Appendix VI.
6.4.5: Contigs of A.annua and A.dubia do not produce homology
The reason why so many genes did not show homology is not altogether
surprising using the nr database. If EST database is used many Expressed sequence
Tags are available for Artemisia. The fact is we still do not know what a lot of genes
do and hence gene functions of many newly sequenced genes of Artemisia can also
not be predicted.
Out of 500 contigs, 244 contigs did not show any homology. However, if any
gene sequence of A. annua and A. dubia does not match with gene sequence in any
data base it is justified if the sequence has completely been changed by insertional and
deletional mutagenesis, during the slow process of evolution. Fast sequence
divergence can cause a sequence to completely change from what was handed down
by the common ancestor. Such gene sequences lose homology.
6.5: Conclusion
In this part, the transcriptome of Artemisia annua and Artemisia dubia
were sequenced for the first time. Gene contigs were generated by alignment of the
sequences derived from a compilation of all the sequences obtained from a species.
The different individual samples from both Artemisia annua and Artemisia dubia
were then compared against this reference gene list to evaluate if any genes in the
genome of the transformed plants are up or down regulated compared to
untransformed plants.
Genes of transgenic lines of Artemisia annua and Artemisia dubia were
found to be highly up regulated as compared to control plants.
Transgenic lines carrying two copy numbers were found up regulated
compared to transgenic lines having one copy number.
Transformed and untransformed plants of A.annua showed up regulation
compared with transformed and untransformed plants of A.dubia.
BLAST of different contigs also showed many enzymes that have
established role in A.annua and A. dubia and were common to various
other species.
These results can have many implications and can be extrapolated to
understand different unknown aspects for a newly sequenced genome.
Chapter No. 7
Discussion
DISCUSSION
Throughout the history plants have been of great importance to medicine.
Plants with medicinal properties have been utilized successfully in the treatment of
ailments of varying degrees of severity. The compounds that are responsible for
medicinal property of the drugs are usually secondary metabolites (Vormfelde, and
Paser, 2000). The use of the medicinal plants for curing disease has been documented
in history of all civilizations (Street, 1977). One such genus is Artemisia; different
species of Artemisia are famous for their medicinal properties like Artemisia dubia
and Artemisia annua. Artemisia species produce a very effective antimalarial
compound artemisinin, which also showed activities against cancer cells,
schistosomiasis, and certain viruses, i.e., human cytomegalovirus, hepatitis B and C
virus, and bovine viral diarrhea virus. Interestingly, the antimicrobial bioactivity of
artemisinin is not just against Plasmodium but it seems to be even broader and also
includes the inhibition of other protozoan such as Leishmania, Trypanosoma, and
Toxoplasma gondii, as well as some trematodes, fungi, yeast, and bacteria. The
analysis of its complete profile of pharmacological activities, as well as the
elucidation of molecular modes of action and the performance of clinical trials, will
further elucidate the full potential of this versatile weapon from nature against
diseases (Efferth, 2009).
Since the discovery and elucidation of chemical structure of artemisinin,
several teams reported different pathways for the synthesis of artemisinin, but all of
them had many steps and a low overall yield. None of these complex synthetic
pathways provide a feasible method for the large-scale production of artemisinin. At
the moment, its extraction from Artemisia species plants remains the only commercial
source for the drug (Avery et al., 2003) and it its concentration varies from 0.01 to 1.5
% DW (Christen and Veuthey, 2001; Ferreira et al., 2005).
Over the past decade, novel features of plant cells transformed with the rol A,
B and C genes have been revealed, such as increased production of secondary
metabolites. The rol A gene has emerged as a stimulator of growth and secondary
metabolism (Altvorst et al., 1992; Schmulling et al., 1993). The rol B protein on the
other hand, has been shown to have a tyrosine phosphatase activity and therefore a
possible role in the auxin signal transduction pathway (Filippini et al., 1996). Estruch
et al. (1999) have demonstrated that rol C can be involved in the release of active
cytokinins from their inactive glucosides due to its cytokinin glucosidase activity and
therefore involved in activation of secondary metabolism. Collectively, these genes
play a major role in the pathway that leads to high levels of secondary metabolites.
Production of transgenic plants seems to be the most appropriate choice to improve
production of secondary metabolites of any plant. For this purpose Agrobacterium
mediated transformation method is being used for several medicinal plants including
Artemisia species (Nin et al., 1996; Nin et al., 1997; Vergauwe et al., 1998).
The demand of artemisinin is increasing due to its multifunctionality e.g.
overall demand of artemisinin and its precursors for treatment of malaria has
increased from 22,000 treatment courses in 2001 to an estimated 200 million
treatment courses in 2008 (WHO, 2008 update). Therefore, the aim of this project was
to enhance and compare artemisinin production in the plants of Artemisia dubia and
Artemisia annua transformed with rol genes by using Agrobacterium tumefaciens and
Agrobacterium rhizogenes with the non transformed plants. This report also covers
the effect of different factors, such as type of explants, sterilization period and cocultivation period, on transformation efficiency and analysis of anti-cancerous
activities on cancer cell lines and their comparison in transformed and nontransformed plants. In addition to it, this report also evaluates the metabolic pathways
through which these rol genes enhance the production of artemisinin and to analyse
its anticancerous activities. This report also explains transcriptome analysis of
transformed and untransformed plants of Artemisia annua and Artemisia dubia.
7.1: Agrobacterium tumefaciens mediated transformation of Artemisia dubia and
Artemisia annua with rol genes
Agrbacterium mediated transformation is an effective and widely used
approach to introduce foreign DNA into dicotyledons plants. For different plant
species different gene transfer protocol are applicable. Various researchers have
reported transformation of Artemisia species with Agrobacterium but mostly they
transformed Artemisia annua. (Biswajit et al., 2000; Vergauwe et al., 1998;
Vergauwe et al., 2002a, Vergauwe et al., 2005). Little work has been done on
Artemisia dubia (Jun-Li et al., 2005, Kaul et al., 1976; Zafar et al., 1990; Nin et al.,
1996; 1997; Rizwana et al., 2002, Altvorst et al., 1992, Charles et al., 1990, Davioud
et al., 1988, Giri and Narasu, 2000). Before the transformation protocol established by
Vergauwe et al, (1996b) no successful procedure had been developed to regenerate
Artemisia plants in short time. Mannan et al, (2008) transformed the Artemisia dubia
with Agrobacterium rhizogenes and obtained transformed regenerated plants and in
1998, Vergauwe studied some more important factors that can affect the
transformation efficiency. The results from these studies were combined to produce
an enhanced protocol for the transformation with Agrobacterium tumefacienes of
Artemisia dubia L. The protocol includes incubation of the explants for five minutes
in Agrobacterium solution, containing 50mg/L kanamycin, having a pH of 5.8 and an
optical density of 1 at 560nm. It was followed by a three days cocultivation period on
MS medium supplied with 200 µM acetosyringone and 50mg/l kanamycin. Explants
were then transferred to selective regeneration medium containing 0.1 mg/L BAP,
50mg/l cefotaxime and 20 mg/L kanamycin. Rooting was obtained on half MS
medium containing 0.025mg/L NAA.
A large number of literatures around the globe has come in print on the
transformation of Artemisia annua, but no work has been done on transformation by
Agrobacterium in Artemisia annua growing naturally in Pakistan. This report explains
an enhanced and efficient protocol for the transformation with Agrobacterium
tumefacienes
and Agrobacterium rhizogenes
of Artemisia annua
L. The
Agrobacterium tumefacienes mediated transformation protocol includes incubation of
the explants for five minutes in Agrobacterium solution, containing 50mg/L
kanamycin, having a pH of 5.8 and an optical density of 1 at 560nm. Three days
cocultivation period was given on MS medium supplied with 200 µM acetosyringone
and 50mg/l kanamycin. Explants were then transferred to selective regeneration
medium containing 1 mg/L BAP, 50mg/l cefotaxime and 15 mg/L kanamycin.
Rooting was obtained on MS medium containing 0.1mg/L NAA.
During
Agrobacterium
tumefaciens
mediated
transformation,
proper
sterilization procedure is required for in vitro germination of seeds. Mercuric chloride
was used as a sterilizing agent. Sterilizing agents play important role in the
germination of seeds, long duration had an inhibitory effect on seed germination and
the seeds became dead while in short time treatment the percentage germination of
seeds increased with minimum germination period. Two minutes treatment was found
to be the most suitable duration for the sterilization of seeds (Fig. 2.2).
Explants from in vitro grown seedlings of 20-days old seedlings were used for
the transformation procedure. Explants were precultured on MS medium for three
days. Co-cultivation period affects the transformation efficiency. MS medium
(Murashige and Sakoog, 1962) containing 200 µM acetosyringone was used as
cocultivation medium and after cocultivation, explants were placed on cocultivation
medium for 2-days. Schmid et al. (2005) showed that this treatment increases the
transformation efficiency up to 20%. It seems that after cocultivation of explants with
Agrobacterium, a certain period of time prior to the shifting of explants to the
selection medium positively influences the transformation efficiency. It probably
allows the T-DNA transfer, integration, transcription and sufficient enzyme
production leading to the expression of kanamycin resistant phenotypes.
In order to select the suitable types of explants for regeneration, different type of
explants were regenerated on media in order to check their regeneration efficiencies.
Leaf and stem explants were used on MS medium containing 0.1mg/l BAP, 20mg/l
kanamycin and 500mg/l cefotaxime. Maximum regeneration efficiency was observed
by stem explants on this medium. So this medium was used as selection medium after
the cocultivation of explants.
After the infection with Agrobacterium, explants were placed on cocultivation
medium for 2-days. Then these explants were shifted to selection medium. In
selection medium 50mg/l kanamycin was used for the selection of transformed
explants as used by Baroncelli et al. (1992), Jorsobe et al. (2003) and Vatsya et al.
(2002). Explants infected with Agrobacterium tumefaciens strain LBA 4404 with
plasmid RT99 and harbouring rol ABC genes. Among the two types of explants used,
stem explants showed maximum transformation efficiency on this SM (MS medium
containing 0.1mg/l BAP, 50mg/l kanamycin and 500mg/l cefotaxime). Leaf explants
were not found to be much efficient in generating transgenic shoots and most of these
died on the selection medium. Thus it appears that leaf explants are not much worth
carrying out transformation experiments. This increased transformation efficiency of
stem explants as compared to leaf explants has also been reported by Skoog and
Miller (1997), Robertson and Earle (2000), Murata (1998), Gamborg et al. (1968) and
Bidney et al. (1991).
For the selection of transformed explants 50mg/L kanamycin and 500mg/l
cefotaxime were used in the selection media. Various reports (Davioud et al., 1988,
Maurel et al., 1991, Morgan et al., 2004) have shown that 50mg/l kanamycin is
sufficient for the selection of transformed plants. The control of Agrobacterium
growth at 500mg/l of cefotaxime has been shown by Vatsya et al. (2002), Jorsobe et
al. (2003) and Bettini et al. (2003).
For determination of best explants for shoot regeneration, different types of
explants were tried. Considering the different kinds of explants, the use of stem
explants seems best for the formation of shoots as well as production of Artemisia
dubia and Artemisia annua plants. Shoot regeneration response observed on MS
medium containing BAP 0.1mg/L, 50mg/l Kanamycin and 500mg/l cefotaxime with
stem explants was 80-100%. Shoot regeneration response of leaf explants was not
good as the leaves mostly formed calli on this medium. It is in accordance with
Schmid et al, (2005) who worked on A. annua and reported that within three weeks
all stem explants of A. annua developed shoots. However the shoot regeneration
continued further and one explants led to the formation of clusters of several shoots,
so that the number of regenerated shoots was higher than the original number of stem
explants (Schmid et al., 2005). Nair et al, (2001) reported the regeneration of
Artemisia plants starting from leaf explants by using a hormone combination of the
auxin NAA (0.05) mg/L and the cytokinin BAP (0.1 to 0.2 mg/L). In about 60 days
distinct shoots were obtained in this procedure, but the rooting of the shoots took 3
months, suggesting that an optimum concentration of hormone in the regeneration
medium highly affects the time of regeneration of explants. The BAP (0.1mg/L) was
found to be best for regeneration of shoots.
Once the transformed shoots were obtained, they were rooted on rooting
medium. In case of Artemisia dubia it occurred by using 0.025mg/L NAA in the
rooting medium and in case of Artemisia annua it occurred by using 0.1mg/L NAA in
the rooting medium, this is in contrary to Vergauwe et al., (1998), who while working
on the Agrobacterium tumefaciens-mediated transformation of A. annua reported high
efficiencies of root induction of shoots grown on non-selective medium. Their study
suggested that cocultivation of the explants with Agrobacterium tumefaciens did not
affect rooting of regenerated shoots. Whereas we have faced great difficulty as both
the transformed and untransformed shoots were not giving roots on the proposed
medium of i.e. 0.1mg/L NAA. However, rooting of Artemisia annua and Artemisia
dubia is in accordance to the results of Jun-Li et al., (2005) and Nair et al., (2001)
who were working with Agrobacterium tumefacienes mediated transformation of
Artemisia annua and found rooting of Artemisia annua on 0.05-2.0mg/L NAA and
0.1mg/L NAA. We got rooting within 1-2 weeks in transformed and untransformed
shooted explants by culturing them on rooting medium containing 0.025mg/L in case
of Artemisia dubia and 0.1mg/L NAA in case of Artemisia annua. According to our
results the number of roots developed on transformed shoots was restricted whereas
Nin et al., (2004), while working on transformed Artemisia annua in vitro grown
plants exhibited a more developed rooting system and roots appeared on the stem too.
These differences could be due to the difference in the specie or more pronouncedly it
is because they had used Agrobacterium rhizogenes for infection and as we know that
it is a natural plant pathogen responsible for adventitious root formation at the site of
infection (Hooykaas, 2004). While we have used Agrobacterium tumefaciens, which
is a soil bacterium and causes crown gall disease (Brown, 2001).
Other factor studied that could affect the transformation efficiency was
cocultivation period. Time of incubation of explants with Agrobacterium inoculums is
an important factor affecting the transformation efficiency. An incubation time of five
minutes was found to be best giving 70% transformation efficiency. Increase in
incubation time led to decrease in transformation efficiency because of over growth of
Agrobacterium that hinders the growth of the transformants and hence has a negative
effect on transformation efficiency. Transformation efficiency is also influenced by
cocultivation time. Explants were cocultivated for two or three days. The highest
formation of transgenic plants was generally obtained after two days of cocultivation.
All the leaf and stem explants showed positive gene expression. It is in agreement
with the results of Vergauwe et al. (1998) as they did cocultivation of Artemisia
annua for 48 to 60 hours. According to their findings longer cocultivation periods did
not help the transformation efficiency because of over growth of Agrobacterium as
mentioned earlier.
For successful Agrobacterium transformation, elimination of bacteria from
culture soon after transformation is necessary. This is achieved by the addition of
antibiotics into the culture medium. Antibiotics, which are commonly used to
eliminate Agrobacterium tumefaciens, have been shown to influence the
morphogenesis either positively or negatively (Jun-Li et al., 2005). Different doses of
cefotaxime were used to optimize the standard dose which can control the overgrowth
of Agrobacterium and also do not inhibit the regeneration. Cefotaxime at a
concentration of 500mg/L controlled overgrowth of bacterium while doses higher
than 500mg/L resulted in necrosis and hence no regeneration was observed. Even
though this concentration of cefotaxime retarded the callus formation, regenerated
plants were able to grow successfully. This is an agreement with Vergauwe et al,
(1996b), who used cefotaxime as decontaminating antibiotic and reported that it
results in retardation of callus formation and inhibition of the shoot inducing capacity
in Artemisia annua.
We have reported some morphological differences that existed among some
transgenic and non-transgenic plants. Increased plant height, broad leaves, branched
and hard stem are some of the features observed among transgenic plants (Table: 2.2).
Such features were not found in control plants and hence seem to be associated with
rol genes as increased plant height and broad leaves have been reported earlier by
Mercke et al. (2000) and Wallaart et al. (2001). These morphological changes could
be because of the hormonal imbalance caused by the rol genes as the rol genes have
been reported to cause functional imbalance in the auxin/cytokinin ratio in favour of
cytokinins (Schmulling et al., 1993).
Regenerated plantlets were potted in the greenhouse, and their survival rate
was 98%, which is in accordance with the findings of Fazal et al, (1997). They
acclimatized the Artemisia absinthium and 95% of the plantlets were successfully
transplanted to soil and continued to grow in the field.
7.2: Agrobacterium rhizogenes mediated transformation of Artemisia dubia and
Artemisia annua with rol genes
Hairy roots produced as a result of genetic transformation by A. rhizogenes,
often grow as fast as or faster than plant cell cultures (Flores and Filner, 1985). The
neoplastic roots are characterized by a high growth rate and are able to synthesize
some secondary metabolites (Flores and Filner, 1985). The greatest advantage of
hairy roots is that hairy root cultures often exhibit about the same or greater
biosynthetic capacity for secondary metabolite production compared to their mother
plants (Banerjee et al., 1998). Even in cases where secondary metabolites accumulate
only in the aerial part of an intact plant, hairy root cultures have been shown to
accumulate the metabolites. For example, lawsone normally accumulates only in the
aerial part of the plant, but their hairy roots can produce lawsone (Bakkali et al.,
1997). Hairy roots many provide the mean to produce these commercially valuable
products on a large scale (Flores and Curtis, 1992; Wilson, 1997).
Little work has been published about the transformation of A.dubia and A.annua
with Agrobacterium rhizogenes growing naturally in Pakistan except one report by
Mannan et al., (2008) transformed the A.dubia with Agrobacterium rhizogenes and
generated hairy roots. The results from these studies were combined to produce an
enhanced protocol for the transformation with Agrobacterium rhizogenes of Artemisia
dubia WALL. and Artemisia annua L. The protocol includes incubation of twenty
eight days old sterile plantlets of A. annua and Artemisia dubia. A single colony of A.
rhizogenes, 24 hours incubated Agrobacterium cultures, was picked with sterile
scalpel which lightly incised into the stem portion of A. annua. In each plantlet, 2-3
incisions were made on different places along the length of stem, by Agrobacterium
rinsed scalpel. The infected plantlets were incubated again in growth room having 24
μM m-2 sec-1 cool white fluorescent light and 25ºC with 16/8 light period. After
seven days of infection, hairy roots started emerging from Agrobacterium infected
stem portions of A. annua and Artemisia dubia. In the present investigation, after
transformation the hairy roots from both Artemisia species and roots from nontransformed plants were shifted to solid B5 medium for further proliferation, the
transformed roots grow faster as compared to non transformed roots. Norma and Ana
Maria (1995) also reported that hairy root culture can significantly increase 2.5-3 fold
the secondary metabolites in comparison with control and also reported that
transformed and non-transformed roots exhibit a strong tendency towards
proliferation in liquid B5 medium.
For transformation with Agrobacterium rhizogenes, small stem (2-4cm)
portions of Artemisia dubia and Artemisia annua having a bud were transferred to MS
medium (Murashige and Skoog, 1962) after sterilization. New plants appeared from
bud after one month and hairy roots appeared in six days after infection with
Agrobacterium rhizogenes strain LBA9402 (Fig. 2.11), while Agrobacterium
rhizogenes strain LBA8196 did not produce any hairy roots. Mannan et al. (2008)
also reported the production of hairy roots in Artemisia dubia and Artemisia indica
with Agrobacterium rhizogenes strain LBA9402 and LBA8196. Nin et al (1997) also
infected different genotypes of Artemisia absinthium shoots with two different
Agrobacterium rhizogenes strain LBA1855 and LBA9402 and reported production of
hairy roots with strain LBA9402 and LBA1855.
Similarly, Giri and Narasu (2001) transformed Artemisia annua with different
Agrobacterium rhizogenes strains such as A4, K599, LBA9402, 9365, 9340 and
reported variable virulency among these strains for induction of hairy roots with the
best transformation response obtained with LBA9402. These reports are in agreement
with our results that LBA9402 is more effective in generating Artemisia dubia and
Artemisia annua hairy roots as compared to LBA8196. Even though LBA8196 did
not produce any transformed roots in Artemisia dubia and Artemisia annua yet the
possibility of this strain to produce transformants in other species of Artemisia cannot
be excluded.
The transformation of Artemisia dubia and Artemisia annua was confirmed by
PCR and Southern blot analysis. All the transformants tested gave positive PCR
results for the rol A, B and C genes. Southern blots also detected the copy number in
independent transgenic lines with the single T-DNA insertion being the most frequent.
Han et al (2005) and Zhang et al (2009) also reported the optimization of
Agrobacterium transformation systems in Artemisia annua and confirmed the
integration of the target genes by Southern blot analysis and found double number in
90% plants. Waleerat et al (2010) and Hobbs et al (1993) also described the
transformation of Artemisia annua with Agrobacterium tumefacienes and confirmed
integration of target genes by southern blot analysis and found double number only in
one plant out of 7 plants. Nin et al (1997) explained the transformation of Artemisia
absinthium with Agrobacterium rhizogenes and confirmed the transformation through
Southern blot analysis and did not find double copy number in any of transformed
plant.
7.3: Analysis of artemisinin and its derivatives
Extraction of artemisinin, artemether, arteether, artesunate and dihydroartemisinin with Ethyl acetate (Ferreira and Janick, 2002), acetonitrile (Elsohly et al.,
1987), chloroform (Woerdenbag et al., 1991; Mary et al., 1994) and petroleum ether
(Klayman et al., 1984) has been reported with the extraction time ranging from a few
minutes (Duke et al., 1994) to several hours (Charles et al., 1990). Charles et al.
(1990) reported rapid extraction and 48 hours long extraction methods of artemisinin
and its derivatives and found no significant difference. In the present project,
extraction of artemisinin from plant material was carried out using Ethyl acetate and
hexane in the ratio of 95:5 and acetonitrile 100% as it is most widely used laboratory
techniques today (Van Quekelberghe et al., 2008; Yan Liu et al., 2009 ; Alexei et al.,
2010). It is worth mentioning here that some alteration in artemisinin and its
derivatives concentration in previously reported work may be possible due to different
methods of extraction. Mingfu et al. (2005) used 50 ml methanol to extract
artemisinin from 2 g of A. annua leaves powder followed by Sonication for 45
minutes and centrifugation at 12000 rpm for 3 minutes resulting in 0.03 to 0.71 %
artemisinin. Elsohly et al. (1987) dried 10 g of A. annua leaves in an oven at 40 ºC for
24 hours, then crushed and extracted with hexane (100 ml) by boiling for 15 minutes
under reflux resulting in 0.153 % artemisinin.
Marcel et al. (1997) compared supercritical fluid extraction (liquid carbon
dioxide and ethanol in a ratio of (97:3)) resulting in 0.3% artemisinin and liquid solid
extraction by ethyl acetate and found that extraction with ethyl acetate give more
artemisinin (0.88 %). Parmeshwari and Ram, (1998) extracted artemisinin from 1.0 g
of dried A. annua leaves in 15 ml hexane for 3 minutes at 40 ºC (0.009 %
artemisinin). Kim et al. (2001) extracted 1.0 g roots of A. annua in 3 ml of ethyl
acetate and hexane for 30 minutes at room temperature (0.003 % artemisinin). Guo et
al. (2005) extracted 5 g of dried A. annua leaves in a Soxhlet extractor with 200 ml
petroleum ether at 60 ºC for 6 hours (0.652 % artemisinin). Congyue et al. (2006)
refluxed 0.5 g dried A. annua leaves in 50 ml of hexane at 75 ºC for 1 hour (0.0226 to
0.785 % artemisinin). Filip et al. (2006) has done extraction by immersing 1.0 gram
of fresh plant material in 6 ml chloroform for one minute evaluating an artemisinin
content of 0.068%.
In the present study, extraction of artemether, arteether, artesunate and dihydroartemisinin from plant material was carried out using ethyl acetate and hexane in the
ratio of 95:5 and acetonitrile 100% as it is most widely used laboratory techniques
today (Van Quekelberghe et al., 2008; Yan Liu et al., 2009 ; Alexei et al., 2010).
Ferreira et al., (1994) extracted artemisinin and its derivatives by refluxing 0.5 g of
sieved dry leaves with 50 ml of hexane at 75 ◦C for 1 h. Artemether acid was
extracted by many methods including extraction with hexane (Kim et al., 2001;
Ferreira et al., 1994), chloroform (Mary et al., 1994) and with supercritical fluid
composed of carbon dioxide and 3% methanol with temperature and pressure fixed at
50 degrees C and 15 MPa, respectively (Kohler et al., 1997). Arteether, artesunate and
dihydro-artemisinin have been extracted mainly through hexane (Ferreira et al.,
1994), Ethyl acetate (Ferreira and Janick, 2002), acetonitrile (Elsohly et al., 1987) and
with chloroform (Woerdenbag et al., 1991; Mary et al., 1994). We used Ethyl Acetate
because it increases solubility, and we use hexane for artemisinin because hexane and
artemisinin both are non-polar, and non-polar dissolves in non-polar, when we add
little amount of ethyl acetate into hexane, it increases the solubility of artemisinin
Many analytical procedures to identify and quantify artemisinin, its biosynthetic
precursors as well as its metabolites have been developed during the last 30 years.
These include thin layer chromatography (TLC) (Klayman et al., 1984), High
performance liquid chromatography with UV detection (HPLC-UV) (Zhao and Zeng,
1985), gas chromatography coupled to mass spectrometry (GC-MS) (Woerdenbag et
al., 1991), capillary electrophoresis coupled to UV detector (CE-UV) (Christen and
Veuthey, 2001) and enzyme-linked immunosorbent assay (ELIZA) (Jaziri et al.,
1993). We successfully used HPLC equipped with auto-sampler, degasser and a
photo-diode array detector method for rapid, accurate and cost effective detection and
quantification of artemisinin and its derivatives. Now days, this is most commonly
used method of many research groups for assay of artemisinin (Towler and weathers,
2007; Kim et al., 2001). Zhou et al. (1988) reported an HPLC method with
polarographic detection of artemisinin and its derivatives. Several groups, (Sandrenan
et al. (1997), Karbwang et al. (1997), Navaratnam et al. (1997), Bangchang et al.,
(1998) developed an analytical method for the determination of artemether, arteether,
dihydro-artemisinin and artesunate based on HPLC and ECD in reductive. HPLC with
evaporative light scattering detection (HPLC–ELSD) (Kohler et al., 1997; Christen
and Veuthey, 2001), gas chromatography with mass spectrometric detection (GC–
MS) (Woerdenbag et al., 1991), GC with flame ionization detection (GC–FID)
(Ferreira et al., 1994; Sipahimalani et al., 1991) and enzyme-linked immunosorbent
assay (ELISA) (Jaziri et al., 1993) are commonly used for analysis of artemether,
arteether, artesunate and dihydro-artemisinin.
Artemisinin, artemether, arteether, artesunate and dihydro-artemisinin analysis
was carried out on dried plant parts (shoots, roots and hairy roots) of transformed and
non-transformed Artemisia annua and Artemisia dubia for the present study. Similar
type of dried plant parts of Artemisia species were used by a large number of other
researchers for analysis of artemisinin and its derivatives (Liu et al., 1979; Acton et
al., 1985; Klayman, 1985; ElSohly et al., 1987; Luo and Shen, 1987; Singh et al.,
1988; Charles et al., 1990; Delabays et al., 1993; Woerdenbag et al., 1994; Marcel et
al., 1997; Parmeshwari and Ram, 1998; Aryanti et al., 2001; Christen and Veuthey,
2001; Delabays et al., 2001; Guo et al., 2005; Mingfu et al., 2005; Arab et al., 2006;
Congyue et al., 2006; Towler and Weathers, 2007).
A prominent feature of our research work is to screen out both transformed and
non-transformed Artemisia species for the presence of artemisinin and its derivatives
by using same conditions of extraction (using ethyl acetate and hexane in the ratio of
95:5 and 100% acetonitrile), same conditions and equipment for analysis (HPLC
equipped with auto-sampler, degasser and a photo-diode array detector) and same
type of plant parts (dried plant parts). Complete data were analyzed by using same
type of software (ANOVA). Therefore, all comparative results can be considered
highly reliable and reproducible.
Artemisinin analysis was carried out on dried plant material (shoots, roots and
hairy roots) of transformed and non-transformed plants of A. annua and A. dubia. The
tissue assayed was similar to that from Artemisia species in previous studies
(Delabays et al.,2001; Guo et al., 2005; Mingfu et al., 2005; Arab et al., 2006;
Congyue et al., 2006; Towler and Weathers, 2007). Our control plants contained
similar amounts of artemisinin when compared to these studies on the vegetative
stage. We found 0.4% DW artemisinin in control plants of A.annua and 0.01 % DW
artemisinin in A.dubia, while Wallaart (1999) et al., found 0.1-0.5% DW artemisinin
in control plants of A.annua, Weathers et al., (2007) found 0.2 -0.5% artemisinin in
vegetative leaves and Mannan et al., (2010) found 0.44% DW artemisinin in A.annua
leaves and 0.05% DW in leaves of A.dubia.
Transformed shoots, roots of transformed shoots and hairy roots of A. annua and
A. dubia all contained significantly more artemisinin when compared to the controls.
The highest concentration of artemisinin was observed in transgenic leaves of both
Artemisia species (i.e. ~ 10 and ~ 30 fold increase in leaves of A. annua and A. dubia
respectively compared to controls). Other researchers have previously reported that
transformation of different Artemisia species results in an increase in production of
artemisinin within the plant. Vergauwe et al., (1998) reported that Agrobacterium
tumefacienes transformation with EHA101 (pTJK136) strain and C58C1RifR
(pGV2260) (pGSJ780BAT) strain of A. annua resulted in an increase in artemisinin
content (0.42%) and its metabolites. Another report by He et al., (1983) reported
0.36% increase in artemisinin amount in leaves transformed with ADS gene of
artemisinin biosynthetic pathway of A. annua as compared to non-transformed plants.
Similarly, Abdin et al. (2003) reported the presence of artemisinin, ranging from 0.01
% to 0.80 %, in leaves and flowers of transformed A. annua and by overexpression of
genes coding for enzymes associated with the rate limiting steps of artemisinin
biosynthesis pathway. Han et al. (2006) reported that artemisinin production increased
by up regulation of FDS through genetic transformation of Artemisia annua with
different genes. Chen et al. (1999, 2000) described that Overexpression of Gossypium
arboretum FPS in hairy roots and transgenic plants resulted in three to four fold and
two to three folds increased in artemisinin content respectively. The results achieved
in this study are significantly higher with increase of 12 fold or greater being detected
depending on the tissue examined.
A meaningful increase in artemisinin content was observed in hairy roots and
roots of transformed shoots of A.annua and A.dubia as compared to control. Similarly
roots of transformed plants in both Artemisia species also showed significant increase
in artemisinin content compared to control roots. An earlier report shows that A.
rhizogenes mediated transformation increased the level of secondary metabolites in
plants (Kovalenko et al., 2004). Artemisinin production (0.42%) has been reported in
hairy roots culture of A. annua (Weathers et al., 1994). Mannan et al., (2008) reported
the production of artemisinin by hairy roots in Artemisia dubia (0.603%) and
Artemisia indica (0.0026%) with Agrobacterium rhizogenes strain LBA9402.
Weathers et al., (2005) reported a significant increase in artemisinin content in hairy
roots culture of A.annua. The concentration of artemisinin in roots of untransformed
A.annua and A.dubia is almost negligible (Figure 3.3). These findings agree with
those of Kim et al. (2001) and Ferreira and Janick, (1996), who found very low
artemisinin concentrations (0.003 %) in root of A. annua. Other studies did not detect
artemisinin in roots of A. annua perhaps due to the sensitivity of the analysis carried
out (He et al., 1983; Kudakasseril, 1987; Fulzele et al., 1991; Woerdenbag et al.,
1991; Kim and Kim, 1992; Gulati et al., 1996). We also found low (A.annua) or zero
(A.dubia) artemisinin concentration in untransformed roots. Transformed plants, hairy
roots and roots of transformed shoots of A.dubia and A.annua were all found to
contain significantly higher amounts of artemisinin.
Our results clearly show that the roots of transformed plants and hairy roots
both contain around 1.5% artemisinin. This is only slightly less than that found in
leaves of A.annua and significantly higher than that found in leaf material from
A.dubia. It has been reported that exogenous artemisinin is capable of inhibiting root
growth at concentrations greater than 10 µg ml-1 (Arsenault et al., 2010). Our results
showed that hairy roots were capable of producing more than 15ug g-1 DW. This
would suggest that there are certain cells within the roots which are acting as storage
compartments for the artemisinin produced. Mannan et al. (2010), has shown that
roots of Artemisia plants are capable of producing artemisinin. Interestingly the roots
do not contain trichomes; hair cells are however present and it is possible that these
cells are also involved in the production and storage of artemisinin. The number of
hair cells also appeared to be present in greater abundance on the leaves of
transformed plants (Figure 4.2B).
Artemether, arteether, artesunate and dihydro-artemisinin analysis was carried
out on dried plant material (shoots, roots and hairy roots) of transformed and nontransformed plants of A. annua and A. dubia. The tissue assayed was similar to that
from Artemisia species in previous studies (Delabays et al.,2001; Guo et al., 2005;
Mingfu et al., 2005; Arab et al., 2006; Congyue et al., 2006; Towler and Weathers,
2007). Our control plants contained 0.105% DW of artemether, 0.15% DW of
arteether, 0.104 % DW of artesunate and 0.2% DW of dihydro-artemisinin in control
plants of A.annua and 0.03 % DW of artemether, 0.015 % DW of arteether, 0.013%
DW of artesunate and 0.02% DW of dihydro-artemisinin in A.dubia.
Transformed shoots, roots of transformed shoots and hairy roots of A. annua and
A. dubia all contained significantly more amount of artemether, arteether, artesunate
and dihydro-artemisinin when compared to the controls. The highest concentration of
these artemisinin derivatives were observed in transgenic leaves of both Artemisia
species (i.e. ~ 12, ~ 20, ~ 14, ~ 11 fold increase in artemether, arteether, artesunate
and dihydro-artemisinin respectively in leaves of A. annua, and ~ 20, ~ 90, ~ 60, ~ 50
fold increase in artemether, arteether, artesunate and dihydro-artemisinin respectively
~ 30 fold increase in leaves of A. dubia compared to controls. To the best of our
knowledge, there is no previous report about effect of transformation on enhanced
production of derivatives of artemisinin. This is first report explaining the effect of
transformation on enhanced production of artemether, arteether, artesunate and
dihydro-artemisinin along with increased production of artemisinin in different tissues
of Artemisia annua and Artemisia dubia.
All the peaks in HPLC were confirmed by using spiked sample in each case for
analysis of artemisinin and its derivatives. In some cases like artemisinin peak of
standard was not matched with the sample because of shifting retention time due to
Annua's matrix and that is why we used the spiked sample which showed increase in
the artemisinin peak. This was one of the reasons for spiking.
In general, statistical analysis showed that artemisinin content was enhanced
much more as compared to its derivatives under the effect of transformation with rol
genes and among two Artemisia species, A.annua is the best species for the
production of artemisinin and its derivatives. Considering the tissue types, shoots and
hairy roots are more appropriate for production of artemisinin and its derivatives.
7.4: Analysis of metabolic pathway and trichome development
In the present study, expression of all the genes involved in artemisinin
synthesis pathway were found to be significantly increased in all transgenic lines of
A.dubia and A.annua (Fig.4.1A, 4.1B, 4.1C, 4.1D and 4.1E), which is in clear
agreement with the data obtained for artemisinin content.
Expression of ADS and CYP71AV1, of which the corresponding proteins
catalyze the formation of amorpha-4, 11-diene and its ultimate conversion to
dihydroartemisinic acid, was increased significantly in transformed shoots of A.annua
and A.dubia. Similarly significant differences in expression of these genes were
observed in hairy roots and roots of transformed plants of both A.annua and A.dubia
compared to untransformed roots. The expression of CYP71AVI corresponds to that
found by Olofsson et al., (2011) however the result for the ADS gene differs as they
were unable to show increase in ADS gene expression in hairy roots.
The ALDH1 gene which catalyses the oxidation of artemisinic and
dihydroartemisinic aldehydes was also highly expressed in transformed plants of both
Artemisia species as compared to untransformed plants, i.e. ~ 7 and ~ 5 times more in
transformed leaves of A.annua and A.dubia, respectively, ~ 5times in roots of
transformed shoots of A.annua and in A.dubia this increased from negligible amounts
to significant levels, with similar increases in hairy roots compared to untransformed
roots (Fig.4.1A, 4.1B, 4.1C and 4.1D). Although all the genes studied showed
increased expression in transformed plants and hairy roots, however induction of
CYP71AV1 and ALDH1 was much more compared to the ADS gene.
TFAR1, known to stimulate trichome development and to catalyze
sesquiterpenoid biosynthesis (Lies et al., 2011) showed significantly increased
expression in all transgenic lines of A.annua and A.dubia (Fig. 4.2A and 4.2B). We
also measured the density of trichomes in transformed and non-transformed plants
and found that transformed leaves of A.annua and A.dubia have a higher density of
trichomes compared to leaves of non-transformed plants. Despite close examination
no trichomes were found on roots and hairy roots cultures of either Artemisia species
but they were shown to contain significant amount of artemisinin as shown in Fig.
3.1. Olofsson et al., (2011), found that leaves and flower buds of A.annua produce
more trichomes than other parts of the plant and also found that roots do not have
trichomes. Although the pathway for the synthesis of artemisinin is well known
(Schramek et al., 2010), it has been widely assumed that the trichomes are the only
source of artemisinin production in the plant (Ferreira and Janick, 1995, 1996b; Tellez
et al., 1999; Olsson et al., 2009) despite several papers describing its presence within
roots (Mannan et al, 2010; Jaziri et al., 1995; Nair et al., 1986; Jha et al., 1988).
Lommen et al (2006) reported that the artemisinin content of the plant is associated
with a rise in trichome density but it continued to rise even after a collapse in the
trichome populations. Our results found the trichome density in A. annua is increased
by 10% following transformation however the artemisinin content increased by
approximately 10 times. In A.dubia trichome density was increased by 9% following
transformation and artemisinin content was increased by approximately 30 times
(Fig.4.2A, 4.2B and Fig. 3.1). These results suggest a possible dissociation between
trichome density and artemisinin content. It is unclear if the genes involved in
artemisinin production are expressed elsewhere in the leaves (Duke et al, 1994).
These results also suggest that other genes may involved in trichome development
because TFAR1 gene also showed expression in roots and hairy roots but these did
not produce any trichomes, or TFAR1 may also require some other genes for trichome
development which may be not present in roots and hairy roots.
Despite the work that has been carried out the maximum artemisinin content
that has been achieved through natural selection is approximately 2% (Graham et al,
2010). It has been previously suggested that the plant was unable to support
concentrations higher than this and that the trichomes capacity for storing artemisinin
is limited (Liu et al., 2010). The current study shows that the plants are capable of
producing and storing much higher amounts of artemisinin than previously thought
both in leaves and in roots. This study shows significant amount of artemisinin
production in transformed roots of A.annua and A.dubia and a small amount of
artemisinin in untransformed roots of A.annua. The detection of significantly raised
levels of expression of the genes involved in artemisinin in transformed roots
associated with the detection of significant amounts of artemisinin in these tissues
suggest that synthesis is occurring elsewhere in the plant as suggested by Duke et al.,
(1994). It should now be possible to determine how roots produce artemisinin hence
elucidating how production could be improved.
Baldi and Dixit (2008) and Wang et al. (2010) have reported that artemisinin
production in plants and culture cells is increased after treatment with methyl
Jasmonate; another report by Lies et al. (2011) examined the effect of cytokinin,
gibberelin and jasmonate on artemisinin production. Similarly Patrick et al. (2010)
described the effect of sugars on the production of artemisinin in A.annua. We
transformed A.annua and A.dubia plants with rol ABC genes of A.tumefaciens and
A.rhizogenes which are responsible for enhancing production of secondary
metabolites in plants. The exact mechanism for the action of the rol genes is still far
from clear (Bulgakov, 2008). It has been suggested that they act through the
stimulation of the plants deference response which includes induction of many of the
hormonal pathways. Many secondary metabolites are thought to be have evolved to
protect plants as they expanded to fill different evolutionary niches. It has been
suggested that artemisinin is an example of such a metabolite as it acts to prevent
predation of the plant by herbivores and it is stimulated by pruning prior to harvest
(Duke et al., 1987). This may explain why the transformation with Agrobacterium rol
genes has such a significant effect on the amount of artemisinin produced by the
transformed plants.
The detection of significantly raised levels of expression of the genes involved
in artemisinin biosynthesis in transformed roots can be correlated with the production
of significant amounts of artemisinin in root tissues. This suggests that synthesis is
occurring in tissues other than the trichomes which contradict previous theories and
alter our perception of how and where artemisinin is produced within the plants.
These findings will help to develop better strategies to increase the production of this
valuable therapeutic drug which will in turn allow greater use of it in the
chemotherapy of malaria and other diseases.
7.5: Analysis of transformed and untransformed Artemisia annua and Artemisia
dubia on breast cancer cell lines
Breast cancer is the most frequent type of cancer all over the world and major
source of mortality among women. Schmidt et al., (2007) reported that death rate
from breast cancer is about 38 deaths per 100,000 women, although a great
improvement in breast cancer treatment has been made and decrease in the death rate
is also observed in advanced countries. MCF-7 is a breast cancer cell line isolated
from 69 years old Caucasian women and established by Soule et al. (1973). MCF-7 is
estrogens receptor positive breast cancer cell lines.
To overcome this cancer a constant research addressing more selective natural
inhibitors with low side effects is continued and each discovery adds to the target. It
has been documented that artemisinin inhibits cell proliferation in breast cancer and
other types of cancers (Tan et al., 2011). In the current project, the effect of crude
extracts of transformed and untransformed plants of Artemisia annua and Artemisia
dubia on MCF-7 breast cancer cell lines was studied by using method of Vanicha and
Kanyawim (2006) Sulforhodamine B (SRB) antiproliferative assay.
In the present study maximum proliferation inhibition at 50µg was observed in
hexane extracts of transformed plants of A.annua (98%) and A.dubia (93%)
respectively with lower IC50 values compared to control plants (Fig.5.1A) with higher
IC50 values. Lai and Sing (2006) also reported that artemisinin is very effective
against breast cancer; they treated rats with 0.02 % artemisinin. After 40 weeks the
rats were monitored for breast cancer. Oral artemisinin significantly delayed (p <0.02)
and in some animals prevented breast cancer development, i.e. 57 % of artemisininfed versus 96 % of the controls. Breast tumors in artemisinin rats were fewer (p
<0.02) and smaller in size (p < 0.05) when compared with controls. Efferth (2006)
also explained the effect of artemisinin and its derivatives on several cancer cells and
reported that artemisinin is very effective drug against many types of cancers
especially breast cancer, colon cancer and lung carcinomas without any side effects.
Similarly significant proliferation inhibition was observed in hairy roots of both
A.annua (91-93%) and A.dubia (85%) compared to untransformed roots (Fig. 5.1A).
The aqueous extracts of transformed and untransformed plants of A.dubia and
A.annua were also tested on MCF-7 breast cancer cells and it was found that
transformed plants of both Artemisia species (89% in A.annua and 84% in A.dubia)
and hairy roots (83% of A.annua and 79% of A.dubia) showed more proliferation
inhibition compared to control plants and roots respectively (Fig. 5.1B). Many
researchers (Efferth et al., 2001; Sing and Lai 2001) reported the antitumor and
cytotoxic potential of crude methanolic extract of A. annua and its n-hexane and
aqueous fractions, using potato disc antitumor assay and SRB cytotoxicity assay using
H157 and HT144 human tumor cell lines. They reported that the aqueous fractions
had most significant activity among all the tested samples. The MCF-7 human cancer
cell line was tested and hexane fraction was found show higher proliferation
inhibition of MCF-7 at 50µM concentration suggesting that A. annua extract had
selective inhibitory potential against different cell lines. Furthermore, the compounds
with selective inhibitory potential can be isolated from n-hexane and aqueous
fractions.
Overall results and IC50 values showed that transgenic and non-transgenic
plants of Artemisia annua and Artemisia dubia have significant activity in
antiproliferative assay compared to aqueous extracts of Artemisia annua and
Artemisia dubia plants (Fig. 5.1 ABC). As artemisinin is better dissolved in hexane
than water due to its physico-chemical properties (low thermal and chemical stability
of the endoperoxide function, low polarity and, hence, poor solubility in water and
good solubility in organic solvents) (Lapkin and Plucinski, 2006; Wright et al., 2010).
It confirmed the effect of artemisinin on cancer cells compared to other secondary
metabolites present in transformed and untransformed Artemisia annua and Artemisia
dubia because artemisinin content and anticancer activity showed significant
correlation with each other (Fig 5.2) in case of hexane extraction compared to
aqueous extraction.
Higher proliferation inhibition was observed in transgenic plants and hairy roots
of Artemisia annua as compared to transgenic plants and hairy roots of Artemisia
dubia because A.annua plants produced more artemisinin compared to A.dubia plants
(Fig. 5.1C). Various other researchers have also reported the potential anticancer
properties of artemisinin and its analogs (Beekman et al., 1997, 1998; Chen et al.,
2003, 2004; Efferth et al., 2001; 2002; Efferth and Oesch, 2004; Jeyadevan et al.,
2004; Lee et al., 2000; Li et al., 2001; Mukanganyama et al., 2002; Posner et al.,
1999, 2003, 2004; Reungpatthanapong and Mankhetkorn, 2002; Sadava et al., 2002;
Sun et al., 1992; Woerdenbag et al., 1993, Wu et al., 2001) but at the same time we
cannot skip the affectivity of other compounds present in crude extracts of both
Artemisia species.
Overall these results showed that the Artemisia plants from Pakistan might be a
promising source of pharmacologically active natural products and among these
Artemisia annua might be the most promising plant for further studies as the
extracts/fractions from this plant showed highest and significant activity. This plant
has the widest use in malaria treatment from many years and now for treatment of
cancer among all the tested species of Artemisia. This suggests that the bioactive
compounds isolated from this plant will be safe for human consumption as medicine
and hence splashes our interest for further studies. Therefore, it can be concluded that
Artemisia species had promising potential for further isolation and purification of
cancer chemopreventive and anticancer compounds. Furthermore, anticancerous
activity could be increased with the enhanced production of other secondary
metabolites including artemisinin in rol genes transformed plants of Artemisia annua
and Artemisia dubia.
7.6: Sequence analysis of Artemisia annua and Artemisia dubia and comparison in
transformed and untransformed plants
Different species evolved as a result of speciation event during the course of
evolution (John and Jeroen, 2004; Ohta, 1989). We come across increasingly complex
genomes as we move up the evolutionary tree. There can be several reasons that have
caused the complexity of genome. Genes can be acquired from other species or they
can be duplicated from the existing genes. Duplication of single gene or group
families (Ohta, 2000), entire genome or a single chromosome or part of it can result in
large genome sequences. Duplication of entire genome is the most rapid means of
increasing gene expression (Regis et al., 2009) and number. Sequences that emerged
as a result of recombination within the genome or were carried across the species by
any means lead to divergence of genomes (Posada et al., 2002). As for example,
viruses can integrate themselves in the host genome. They can incorporate their own
genes and also those that they carry across from different species. The fact that
viruses can cross the species barrier makes it even attractive proposition (Linda and
Van, 2009). Replication slippage and DNA amplification (David and Rafael, 1997)
mostly create gene duplication. These evolutionary tools lead to sequence homology
amongst different organisms. Their genomes overlap to varying degree depending
upon the time of divergence. Proteins in different species show shared features.
Consensus sequences give a rational proof of structural and fictional similarity of
proteins.
In this study, the transcriptome of Artemisia annua and Artemisia dubia were
sequenced for the first time. Gene contigs were generated by alignment of the
sequences derived from a compilation of all the sequences obtained from a species.
The different individual samples from both A.annua and A.dubia were then compared
against this reference gene list to evaluate if any genes in the genome of the
transformed plants are up or down regulated compared to untransformed plants. The
Bowtie and CuffLinks programs were used to look for differential expression and
CuffDiff used to derive values. A comparison was also made between A.annua and
A.dubia (Table 6.1) plants. Contigs/genes for which significant differences were
obtained were compared between biological replicates to determine their overall
significance. Those which showed significant differences were submitted to the
BLAST program using both the nr/nt and EST database available at NCBI. The
homology of their genome sequence was found and significant results were obtained.
From the results obtained the putative gene functions could be derived. Furthermore,
genome sequences of individual transgenic lines, which were different from each
other in copy number, were also compared. Besides, genome sequence of
untransformed and transformed A.annua was compared individually with genome
sequence of both untransformed and transformed A.dubia (Table 6.2).
Up regulation and down regulation of different samples was analyzed on the
basis of significant value obtained by generating contigs from different samples
(Table 6.1). Those samples were considered to be highly up regulated that contained
significant value less than 0.01. Transgenic lines of A.annua (A1, A2) and A.dubia
(D1) were compared with control plants (Ac and Dc, respectively). Genes of both
transgenic lines were found to be highly up regulated as compared to control plants.
Although the different transgenic lines produced were inserted with only rol genes,
the whole transcriptome was found upregulated. The reason behind this factor can be
the influence that these rol genes might be able to exert on the expression system of
other coding region of genome. Findings about the over expression of genes that had
different copy number in transgenic lines (A1 and D1 has two copy number, A2 has
single copy number) can be testified. Principally, those genes that have more copy
number, shall exhibit more number of transcripts, conditioning that both copies are
functional to produce transcripts. Another factor can be the modulation of promoter
region in such a way that may have played role in the enhanced expression of gene.
Inserted rol genes construct can integrate in gene sequence to cause Insertional
mutagenesis (Yi Hong, 2008; Regis et al., 2009). It can also interfere with the
promoter region and affect the expression (Renata et al., 2010). The sequence of rol
ABC gene may have provided sites for epigenetic modification that has altered the
DNA sequence to assist the transcription process (Austin and Dixon, 1992; Grønbaek
et al., 2007; Riggs et al., 1996; Portela and Esteller, 2010). The sequence of inserted
rol gene can also play role as an activator or enhancer.
When two transgenic lines of A.annua (A1 and A2) were compared with each
other, transgenic line carrying two copy number (A1) was found up regulated
compared to transgenic line having one copy number (A2). It shows that both the
copies of A1 transgenic line were functional and giving enhanced expression relative
to A2 with single copy of gene confirming the findings of Jun et al., (2011) who
explained that gene expression is directly related to the copy number of genes.
Transformed and untransformed plants of A.annua when compared with
transformed plants of A.dubia were shown upregulated. Up regulation was also
observed when transformed and untransformed plants of A.annua were compared
with control plants of A.dubia. These results show that genome of A.annua is more
prone to epigenetic modifications (Portela and Esteller, 2010). In A.annua Insulator
sequences seem more efficient in molecular assembly on them (Maria et al., 2012)
and are a route of influence to give it an edge for up regulation over transgenic plants
of A.dubia.
It is interesting to note that the pattern of artemisinin production happens to
match with the up regulation of genes of transgenic lines shown in table 6.1. Amount
of artemisinin was found higher in transgenic lines of A.annua as compared to
control. The same pattern follows when gene up regulation is analyzed between
transgenic and control plants (Table 6.1). Similarly, transgenic lines of A.annua and
A.dubia were examined for artemisinin production, transformed A.annua showed
more amount of artemisinin than transgenic line of A.dubia (Graph 3.1). Gene
expression pattern of A.annua and A.dubia transgenic lines goes in harmony with the
artemisinin production in both lines. Same can be observed by analysis of control and
transgenic lines of A.dubia, where transformed samples have more artemisinin
content, a pattern that is similar to gene up regulation in these two lines.
Hence gene up regulation and artemisinin production can be correlated. It can be
said that rol genes affect the genes of artemisinin biosynthesis in a similar fashion as
they do to up regulate other genes. This speculation makes sense because it is
represented by our results of RT-PCR (Fig 4.1 A, B, C, D, and E) that rol genes affect
the genes involved in artemisinin biosynthesis pathway.
BLAST of Various contigs which were obtained by aligning different samples
of A.annua and A.dubia were performed and sequence homology presented interesting
results. These contigs showed homology with different genome sequences ranging
from 60% to 90% but only those Contigs were analyzed that showed 90% homology
or more than it (Table 6.2). Contigs found as a result of aligning A1 and A2 produced
more than 90% homology with different genes of Lactuca sativa, Solanum
lycopersicum, Lycopersicum esculentum, Vitis vinifera and Malus x domestica.
Amongst those genes WRKY 17 which is a key regulatory transcription factor in the
artemisinin biosynthesis pathway (Dongming et al., 2009) was also found with the
highest similarity to Malus x domestica (Apple). Hence it can be suggested that in
Malus x domestica WKRY 17 influences pathway; it is interesting to speculate what
pathway this might be.
Like A1 and A2, contigs from other samples of A.annua and A. dubia (A1 and
AC, A2 and AC, A1 and D1, A2 and D1, AC and D1, A1 and DC, A2 and DC, D1
and DC and AC and DC) also showed sequence homology with different genes of
different genomes present in nr/nt and EST databases of NCBI.
These contigs showed 97%-100% sequence homology mostly with genomes of
Homo sapiens, Mus musculus, Arabidopsis thaliana, Drosophila, Vitis vinifera, Oryza
sativa, Lycopersicum esculentum, Coffea arabica, Solanum lycopersicum, Mediacago
truncatula, Helianthus species, Honey bee, Zebra fish, Zea mays, pig and some EST
sequences of A.annua. This sequence homology of contigs may suggest that these
genes bear functional similarity (Brown, 2002). It is also possible that this
overlapping sequence may code a part of transcript that is common to them (Eugene
and Michael, 2003). It can also mean that the overlapping sequences are
evolutionarily closely related that diverged over the course of time (Koonin, 2005).
The presence of almost 100% sequence homology of contigs from these two
Artemisia species with different animals e.g. Homo sapiens, Mus musculus, Honey
bee, Zebra fish and Pig may represent the presence of any gene that codes for product
which is common in all species. Presence of similar sequences in contemporary plants
and animals support the idea of common progenitor (Maria et al., 1990).
Conclusion
Conclusively, this project represents the comparison of the artemisinin
contents observed in the transformed and untransformed shoots, roots and hairy roots
of Artemisia annua and Artemisia dubia transformed with rol genes through
A.tumefacienes and A.rhizogenes mediated transformation.
Artemisinin was
significantly increased in the transformed shoots, hairy roots and roots of transformed
shoots compared to control in both A.annua and A.dubia plants. Artemether, arteether,
dihydroartemisinin and artesunate contents were also significantly increased in
transgenic shoots, roots and hairy roots to control. Amount found in roots of
transformed shoots and transformed hairy roots was significantly greater compared to
the control roots in which negligible amounts was detected.
Metabolic pathway of artemisinin production in Artemisia annua and
Artemisia dubia and the effect of rol genes through which these genes enhance the
production of artemisinin were analyzed using different genes involved in artemisinin
production and TFAR1 involved in trichome development and sesquiterpenoid
biosynthesis was also studied. Pronounced changes in the expression of the
artemisinin biosynthetic pathway genes were observed in all transgenic lines of A.
annua and A. dubia transformed with rol ABC genes. Expression levels of ADS,
CYP71AV1, ALDH1 and TFAR1 were significantly increased in transformed plants
and hairy roots of both Artemisia species compared to untransformed plants.
Hexane and aqueous extracts from transformed and untransformed plants
of Artemisia annua and Artemisia dubia were analyzed for anticancerous activity
against MCF-7 breast cancer cell lines. Crude extracts of rol genes transgenic plants
revealed higher anticancerous activities against MCF-7 breast cancer cell compared to
control plants. Hexane extracts of rol ABC genes transgenic plants revealed higher
anticancerous activity against MCF-7 breast cancer cell lines compared to aqueous
extracts of plants.
Transcriptome of Artemisia annua and Artemisia dubia were sequenced
for the first time. Genes of transgenic lines of Artemisia annua and Artemisia dubia
were found to be highly up regulated as compared to control plants. Moreover,
transgenic lines carrying two copy numbers were found up regulated compared to
transgenic lines having one copy number. BLAST of different contigs also showed
many enzymes that have established role in A.annua and A. dubia and were common
to various other species. These results can have many implications and can be
extrapolated to understand different unknown aspects for a newly sequenced genome.
Future strategies
Transformation with individual rol A, B and C genes could be carried out to
check their effect on artemisinin production and their comparison with
combined rol ABC genes could be studied.
Further research may be carried out to study the gene expression in T1 and T2
generations by RT-PCR and Northern blot analysis.
Maximum number of Artemisia species could be genetically transformed with
rol ABC genes for increased, constant and permanent source of this valuable
drug.
Artemisinin content could be further increase by transformation with other
genes involved in artemisinin biosynthesis pathway.
Other In-vivo and In-vitro assays may be carried out to unlock other properties
of artemisinin and different Artemisia species.
Sequencing of whole genome of Artemisia may be helpful in predicting the
molecular functions of genes of different Artemisia species.
Stimulation of the synthesis of artemisinin within these plants has also allowed
us to alter our perception of how and where artemisinin is produced within the
plants. These findings will help to develop better strategies to increase the
production of this valuable therapeutic drug which will in turn allow greater
use of it in the chemotherapy of malaria and other diseases.
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Publications
Publications
1. Bushra Hafeez Kiani, Naila Safdar, Abdul Mannan and Bushra Mirza
(2012). Comparative Artemisinin analysis in Artemisia dubia transformed
with two different Agrobacteria harbouring rol ABC genes. POJ. 5(4):386391.
2. Ali M, Kiani BH, Mannan A, Ismail T and Mirza B (2012). Enhanced
production of artemisinin by hairy root cultures of Artemisia dubia.
Journal of Plants Medicinal Research. 6(9): 1619-1622.
3. Bushra Hafeez Kiani, Bushra Mirza and Guy Cameron Barker (2012).
Artemisinin production in Artemisia annua and Artemisia dubia following
transformation with the rol ABC genes and elucidation of the sites of its
synthesis. Plant Physiology. (Submitted)
4. Bushra Hafeez Kiani, Nazif Ullah, Ihsan Ul Haq and Bushra Mirza
(2012). Comparative pharmacological evaluation of rol ABC genes
transformed Artemisia dubia with untransformed plants.
BMC
Biotechnology. (Submitted)
5. Bushra Hafeez Kiani, John Suberu, Guy Cameron Barker and Bushra
Mirza (2013). Development of efficient miniprep transformation methods
for Artemisia annua using Agrobacterium tumefaciens and Agrobacterium
rhizogenes. Plant Breeding. (Submitted)
6. Bushra Hafeez Kiani, Muhammad Tahir Waheed, John Suberu, Guy
Cameron Barker and Bushra Mirza (2012). Anticancer activities of
transformed and untransformed Artemisia annua and determination of
functional compounds. Phytomedicine. (Submitted)
Appendices
APPENDIX-I
YMB medium
K2HPO4
0.5mg/l
MgSO4
2g/l
NaCl
0.1g/l
Mannitol
10g/l
Yeast extracts
10g/l
pH
7.0
APPENDIX-II
MYA Medium
Yeast extracts
Cas-amino acid
5g/l
0.5mg/l
Mannitol
8g/l
NH4 (SO4)2
2g/l
NaCl
5g/l
pH
6.6
APPENDIX-III
Composition of Murashige and Skoog (1962) medium
S. No
Constituents
Formula
Conc. in stock
solutions g/l
Volume of stock/l of
medium (ml)
Macronutrients
1
Potassium nitrate
KNO3
38
2
Ammonium nitrate
NH4NO3
33
3
Calcium chloride
CaCl2.2H2O
8.8
4
Magnesium sulphate
MgSO4.7H2O
7.4
5
Potassium
Phosphate
KH2PO4
3.4
50
Micronutrients
6
Manganese sulphate
MnSO4. H2O
4.4
7
Zinc sulphate
ZnSO4.H2O
1.72
8
Boric acid
H3BO3
1.24
9
Potassium iodide
KI
1.67
10
Sodium molybdate
Na2MoO4.2H2O
0.05
11
Copper Sulphate
CuSO4.2H2O
0.01
12
Cobalt chloride
CoCl2.6H2O
0.005
5
Iron Source
13
Sodium EDTA
Na2EDTA.2H2O
7.46
14
Ferrous sulphate
FeSO4.7H2O
5.56
5
Organic Supplements (Vitamins)
15
Myo-inositol
20
16
Glycine
0.4
17
Nicotinic acid
0.1
5
0.1
18
Pyridoxine-HCl
19
Thiamine-HCl
0.1
Carbon Source
20
30 g /l of Sucrose was used in MS medium.
Appendix IV: For B5 Stock solution preparation
S No
Ingredient Name
Company Name
Stock Name
Concentration g/L
USE
1
Ammonium sulfate
A-4915 (Sigma)
1.01M (NH4)2
SO4
134
1ml/L
2
Boric acid
B-0252 (Sigma)
0.048M H3BO3
3
1ml/L
3
Calcium chloride
dehydrate
Cobalt(II) chloride
hexahydrate
Copper(II) sulfate
pentahydrate
Magnesium sulfate
heptahydrate
Manganese(II)
sulfate
monohydrate
Potassium iodide
C-2536 (Sigma)
1.02M
CaCl2.2H2O
0.1mM
CoCl2.6H2O
0.1mM
CuSO4.5H2O
1.01M
MgSO4.7H2O
0.059M
MnSO4.H2O
150
1ml/L
0.025
1ml/L
0.025
1ml/L
250
1ml/L
10
1ml/L
4
5
6
7
8
9
Ethylenediaminetet
raacetic acid
disodium salt
dehydrate
202185 (Sigma)
209198 (Sigma)
M-7774 (Sigma)
M7899 (Sigma)
207969 (Sigma)
4.5mM KI
0.750
1ml/L
E-5134 (Sigma)
0.11M
Na2EDTA.2H2O
37.3
1ml/L
10
Sodium molybdate
dehydrate
Sodium phosphate
monobasic
dehydrate
Zinc sulfate
heptahydrate
M-1651 (Sigma)
13
14
11
12
15
16
17
1.03mM
Na2MoO4.2H2O
1.08M
NaH2PO4.2H2O
0.25
1ml/L
150
1ml/L
Z-0251 (Sigma)
6.9mM
ZnSO4.7H2O
2
1ml/L
myo-Inositol
I-3011 (Sigma)
9.91
10ml/L
Thiamine
hydrochloride
Pyridoxine
hydrochloride
Nicotinic acid
T-1270 (Sigma)
0.055M MyoInositol
0.03M ThiaminHCl
4.86mM
Pyridoxine-HCl
8mM Nicotinic
Acid
0.1M
FeSO4.7H2O
2.47M KNO3
10
1ml/L
1
1ml/L
1
1ml/L
27.8
10ml/L
249.7
10ml/L
Sucrose
30
30g/L
71505 (Sigma)
P-8666 (Sigma)
N-0765 (Sigma)
F-7002 (Sigma)
18
Ferrous sulphate
heptahydrate
Potassium nitrate
19
Sucrose
S391(Phytotechnolog
y Lab)
P8291 (Sigma)
APPENDIX-V
Southern Blot Solutions
Denaturing Solution (1 liter)
0.5 N NaOH
20.0 g NaOH
1.5 M NaCl
87.7 g NaCl
ddH2O to final volume
Neutralizing Solution (1 liter)
1 M Tris
121.1 g Tris Base
3 M NaCl
175.4 g NaCl
fill to approximately 80% volume with ddH2O
pH to 7.0 with concentrated HCl (≈60 ml/liter)
bring to volume with ddH2O
Prehyb & Hybridization Solution (500 ml)
50% Formamide
250 ml Formamide
3X SSC
75 ml 20X SSC
1X Denhardt‘s Solution
5 ml 100X Denhardt‘s
20 μg/ml salmon sperm DNA
1 ml 10 mg/ml
5% Dextran Sulfate
25 g Dextran Sulfate
2% SDS
100 ml 10% SDS
Distilled water
APPENDIX-VI
Contigs of A.annua and A.dubia showed
80-85% homology
S.No.
Contigs
1
Contig5332:0-407
2
Contig5575:0-732
3
Contig9512:0-1058
4
Contig9144:0-754
5
Contig951:0-251
6
Contig10524:0-757
7
Contig10536:0-983
8
Contig1181:0-4663
9
Contig1260:39-212
10
Contig1320:0-1308
11
Contig1746:0-2322
12
Contig2006:0-880
13
Contig2028:0-994
14
Contig2799:0-245
15
Contig2903:0-2391
16
Contig231:0-650
17
Contig2859:34-864
18
Contig2799:0-245
19
Contig3836:0-1711
20
Contig1972:29-1001
21
Contig231:0-650
22
Contig2622:14-797
23
Contig3934:0-1618
24
Contig981:16-763
25
Contig3437:0-1709
26
Contig4685:0-615
27
Contig5168:6-688
28
Contig7008:0-1433
29
Contig7150:0-1455
30
Contig7416:0-1030
31
Contig10057:0-852
32
Contig1972:29-1001
33
Contig2454:22-1644
34
Contig2799:0-245
35
Contig3437:0-1709
36
Contig7008:0-1433
37
Contig7150:0-1455
38
Contig1907:54-728
39
Contig231:0-650
40
Contig4749:0-1057
41
Contig8881:0-761
42
Contig2698:0-454
43
Contig4685:0-615
44
Contig478:36-283
45
Contig7150:0-1455
46
Contig7015:0-1455
47
Contig2006:0-880
48
Contig2288:0-726
49
Contig2454:22-1644
50
Contig2622:14-797
51
Contig290:0-475
52
Contig318:0-498
53
Contig4168:0-828
54
Contig4704:0-1313
55
Contig5025:0-1950
56
Contig5482:0-902
57
Contig583:0-1445
58
Contig6601:0-889
59
Contig762:0-855
60
Contig8568:0-1291
61
Contig4971:0-638
62
Contig5008:0-2908
63
Contig10081:0-821
64
Contig10271:0-621
65
Contig5821:0-949
66
Contig9460:0-1091
67
Contig9658:53-1094
68
Contig5624:0-2559
69
Contig7019:0-704
70
Contig489:21-1070
71
Contig7255:2-1153
72
Contig7356:0-819
73
Contig3512:0-616
74
Contig10271:0-621
75
Contig10867:0-1043
76
Contig7251:0-1059
77
Contig2139:0-1197
78
Contig359:18-539
79
Contig462:0-927
80
Contig1845:0-590
81
Contig3395:0-424
82
Contig6427:0-816
83
Contig6578:0-492
84
Contig7546:0-614
Bushra Hafeez Kiani