Professional Documents
Culture Documents
Nadeem MSc-Thesis Presentation
Nadeem MSc-Thesis Presentation
Supervisors:
Prof. Dr. Edgar Mai Prof. Dr. Gnther Scherer Prof. Dr. Hartmut Sttzel
Outline
Introduction Objectives Hypotheses Methodology Results Conclusions
Introduction
Plant viruses classification (based on type of genome) DNA RNA
dsRNA ssRNA
Introduction- dsRNA
Partitiviridae and Reoviridae Partitiviridae include three genera:
Partitivirus Alphacryptovirus Betacryptovirus
RdRp CP
VCV RNA1 & RNA 2
DsRNAs with molecular weights of 1.37 x 10 6 Da, 1.26 x 10 6 Da and 1.21 x 10 6 Da. (Abou-Elnasr et al., 1985) Genome consists of dsRNA, encoding RdRp and CP. Contains no movement protein
Introduction
Potato virus X (PVX)
Virions not enveloped 470-580 nm long; 11-13 nm in diameter Symmetry helical Distribution: through contact between the plants subjected to friction
Potato virus X x300,000
www.rothamsted.bbsrc.ac.uk/... www.rothamsted.bbsrc.ac.uk/...
Objectives
Generation of transgenic N. benthamiana expressing RdRp, CP or both Characterization of transgenic plants Evaluation of the susceptibility of transgenic plants to virus infection (PVX) Evaluation of RdRp and CP as silencing suppressors
Hypotheses Statement
Vicia cryptic virus RdRp and/or CP will affect the pathogenic behavior/ pathogenicity of the PVX in N. benthamiana Vicia cryptic virus will increase the symptoms / severity of the PVX RdRp and/or CP act as suppressors of gene silencing
Objective
LB
Kan
pe35S
CP
tnos
RB
LB
Kan
pe35S
RdRp
tnos
pe35S
CP
tnos
RB
One cassettes having both CP & RdRp Not drawn to scale; Marker are omitted
Method- Transformation
Binary vector:pLX222 or pBIN19 Agrobacterium tumefaciens LBA4404 Explants in a petri plate: Size of each explant: 1 cm One transformation = 3 plates = 45 explants
http://www.agriculture.purdue.edu/agbiotech/images/leafdisk1.html http://www.agriculture.purdue.edu/agbiotech/images/leafdisk1.html
Results
Table : Number of calli and T0 plants achieved in 12 transformation experiments
Regenerated T0 plants 34 29
Results
Table: Segregation ratio for germinated seedlings from T0 plants in MS + Kanamycin medium. Chi square values (X) are significant when less than 3.38 at = 0.05.
Green seedling s 80 78 95 31 92
Yellow seedling s 17 19 1 30 4
Experiment 2 Germination and growth comparison of selected transgenic Nicotiana benthamiana lines
Objective
Method
Sowing of seeds 50 seeds in one pot 3 pots per line
Colection of data Counting of germinated seeds after 1st week Shifting seedlings in trays and counting :2nd week Measurement of height and counting of leaves: 3rd, 5th, 7th and 10th weeks
Results
Table: Germiantion of seeds in selected transgenic lines
Results
2,5
1
A A A A A
12
Control CP RdRp Control = 96 CP1 n=115 CP5 n= 124 RdRP1 n=113 RdRp7 n= 84 RdRp8 n= 96
A B
2,0
10
Height (cm)
1,5
Height (cm)
1,0
0,5
2
Plant lines
Plant lines
30
3
C BC D
4
A BC C
A AB
AB
25
50
Control CP RdRp Control n= 25 CP1 n= 25 CP5 n= 25 RdRp1 n=25 RdRp7 n=25 RdRp8 n=25
20
40
Height (cm)
15
Height (cm)
30
10
20
10
Plant lines
Plant lines
Fig : Comparison of plant heights between transgenic N. benthamiana (T1) coat protein lines (CP 1, CP 5) and RNA dependent RNA polymerase lines (RdRp 1; RdRp 7; RdRp 8) with non-transgenic N. benthamiana. 1 = at 3rd week of germination, 2 = at 5th week of gerrmination, 3 = at 7th week of germination and 4 = at 10th week of germination.
Results
Height comparison
50 CP1 CP5 RdRp1 RdRp7 RdRp8 Control
40
Height (cm)
30
20
10
0 3 5 7 10
Weeks
Fig: Comparison of leaf number between transgenic N. benthamiana, coat protein lines (CP 1, CP 5); RNA dependent RNA polymerase lines (RdRp 1, RdRp 7, RdRp 8) with non-transgenic N. benthamiana at 3rd, 5th, 7th and 10th week.
Results
7
1
A AB D CD BC A
20
Control CP RdRp Control n=96 CP1 n=115 CP5 n=124 RdRp1 n=113 RdRp7 n=84 RdRp8 n=96
A B
BC 15 D D
Number of leaves
Number of leaves
CD
10
Plant lines
Plant lines
18 16 14
250
AB
A BC D CD BC
Control CP RdRp
200
4
A B B B
Number of leaves
Number of leaves
150 C
C 100
50
Plant lines
Plant lines
Fig : Comparison of number of leaves between transgenic N. benthamiana (T1) coat protein lines (CP 1, CP 5) and RNA dependent RNA polymerase lines (RdRp 1; RdRp 7; RdRp 8) with non-transgenic N. benthamiana. 1 = at 3rd week of germination, 2 = at 5th week of gerrmination, 3 = at 7th week of germination and 4 = at 10th week of germination.
Results
Number of leaves comparison
250 CP1 CP5 RdRp1 RdRp7 RdRp8 Control
200
Number of leaves
150
100
50
0 2 4 6 8 10 12
Weeks
Fig: Comparison of leaf number between transgenic N. benthamiana, coat protein lines (CP 1, CP 5); RNA dependent RNA polymerase lines (RdRp 1, RdRp 7, RdRp 8) with non-transgenic N. benthamiana at 3rd, 5th and 10th week.
Results
Fresh weight comparison
60 A 50 BC 40 ABC BC AB C Control CP RdRp Control n= 25 CP1 n= 25 CP5 n= 25 RdRp1 n= 25 RdRp7 n= 25 RdRp8 n= 25
Weight (gm)
30
20
10
plant lines
Fig : Comparison of fresh weight between transgenic N. benthamiana, coat protein lines (CP 1, CP 5); RNA dependent RNA polymerase lines (RdRp 1, RdRp 7, RdRp 8) with non-transgenic N. benthamiana.
Objective
Evaluation of PVX interaction with transformed plants having CP and RdRp genes of
Method
Mechanical inoculation
PVX strain: 0018
Five lines were tested by ELISA Observations: 4 and 7 days post inoculation
Results
3,0
2,5
A A
4 DPI 7 DPI
2
A A A
2,5
CP: n = 16 RdRp: n = 24
Absorbance (415 nm)
2,0
Control 1: n = 4 Control 2: n = 4
1,5
Plant lines
Plant lines
0,8
0,7
4 DPI 7 DPI
4
A A A
0,6
0,6
4DPI 7DPI
Control 1: n =4 Control 2: n =4
0,4 a 0,2 a a a
0,2
0,1
0,0 CP RdRp Control1 Control2
Plant lines
Plant lines
Fig : Quantification of PVX in transgenic N. benthamiana CP and RdRp lines at 4 and 7 d.p.i. 1 = experiment 1, 2 = experiment 2, 3 = experiment 3 and 4 = experiment 4.
Objective
httpallserv.rug.ac.be~avierstrprinciplespcr.html
Peter M. Waterhouse NATURE | VOL 411 | 14 JUNE 2001 | www .nature. com
LB
Kan
pe35S
RdRp
tnos
RB
LB
Kan
pe35S
GFP
tnos
RB
LB
Kan
pe35S
HC-Pro
tnos
RB
Fig:
Method
Agroinfiltration:
N. benthamiana (16c line): 5 plants per treatment Agrobacterium tumefaciens: C58C1 Binary vector: pBIN19 Treatments applied:
green colour defuse/ red ring appears green colour persist/ no red ring appears
? ?
Results
GFP
HC-Pro+GFP
CP+GFP
RdRp+GFP
Fig: Effect of CP, RdRp,HC-Pro when applied with GFP and GFP alone in 16c plants.Top pannel 2d.p.i, middle 4 d.p.i and lower 7 d.p.i.
Results
GFP
HC-Pro+GFP
CP+GFP
RdRp+GFP
Fig: Effect of CP, RdRp,HC-Pro when applied with GFP and GFP alone in 16c plants.Top pannel 2d.p.i, middle 4 d.p.i and lower 7 d.p.i.
Conclusions
CP gene interfere while RdRp does not interfere significantly in the growth of N.
benthamiana
Transgenic plants having RdRp and CP gene of VCV are susceptible to PVX like nontransgenic plants
RdRp and CP gene does not act as suppressor for gene silencing
THE END