Embed Size (px)
Citation preview
Internship presentation
Sunmeet Singh Bhatia
Arabidopsis Thaliana
• Thale cress (Mouse-ear Cress).• Model organism – 120-megabase – fully sequenced genome.– 5 chromosomes.– ~20,000 genes.
• Life cycle: 6 weeks
(Boyes et al, 2001; TAIR)
CPR5 (CONSTITUTIVE EXPRESSER OF Pathogenesis-Related GENES 5)
Working with this specific gene in the Arabidopsis genome.
• 2.4 kb gene size• 564 aa long protein • Found on the 5th chromosome
(TAIR)
24-day air grownEarly leaf senescence
cpr5-2 plants
WT
cpr5-2
• ROS generation
HR execution
Resistance: Bacterial & fungal
Aberrant Leaf trichomes
CPR5 mutant abnormalitiescpr5-2WT
Slide Courtesy: Muhammad Faisal
WT cpr5-2
Project
Try to uncouple the different processes of Arabidopsis in which
CPR5 is involved.
How do we do that?
Complementation!
WTcpr5-2
COMPLEMENTATION STRATEGY
Employed strategy for motifs/sites elucidation
Constructs
Wild type phenotype restorationRescue ?
Genotyping
Genotyping the complemented lines to confirm the success of
complementation.
Genotyping
• Genomic DNA
• Genotyping strategy
– PCR using gene specific primers
– Digestion of amplified product
Results- PCR products
1: Col-0 2:cpr5-2 3-25: Complemented lines C: -ve control
Expected amplified product size = 1.468 kb
1.5kb
1.0kb0.8kb0.6kb0.4kb0.2kb
1.5kb
1.0kb0.8kb0.6kb0.4kb0.2kb
Restriction Digest
Restriction enzyme
Restriction enzyme
Restriction enzyme
Restriction enzyme
1 2 3
1
Constructed CPR5 gene
cpr5-2 mutant gene
Restriction site PRESENT
Restriction site ABSENT
Results- Restriction digest• Used BamHI restriction enzyme • Expected BP of fragments 460 bp + 1008 bp
1: Col-0 2:cpr5-2 3-25: Complemented lines
1.5kb1.0kb0.8kb0.6kb0.4kb0.2kb
1.5kb1.0kb0.8kb0.6kb0.4kb0.2kb
Results/Conclusions
Amplification only from complemented linesand
Migration of expected number and size of DNA fragments on gel
confirms successful complementation
Trichomes in Arabidopsis
• CPR5 associated with trichome development(Jing et al., 2008)
• It promotes the growth of trichomes
• cpr5-2 mutants show close to no trichomes
• Col-0 (wild type) has a characteristic pattern for trichomes
(Kirk et al., 2001)
• ‘~WT’; < WT; ‘Partially complemented lines’ & ‘ Not
complemented lines’
Results/conclusions
Col-0 cpr5-2 H-IV: 5-4 (Tray 2)
J-II: 1-1 A-VI: 10-5 A-V: 5-1 H-IV: 1-5 J-II: 2-6 H-IV: 4-5 (Tray 2)
H-IV: 7-4 (Tray 2)
0
20
40
60
80
100
120
140
1-branch 2-branch 3-branch 4-branch
~WT >WT Partially complemented lineNot complemented line
ROS levels in Arabidopsis
• ROS (Reactive oxygen species): bi-product of various metabolic activities of plants
• CPR5 is a master regulator of cellular ROS levels in Arabidopsis (Jing et al, 2008)
• cpr5-2 mutants show higher levels of ROS than the wild-type plant (Jing et al, 2008)
• Based on the ROS levels, complemented lines– Fully complemented (WT-like) – and not-complemented lines (cpr5-2 mutant-like)
• ROS levels (Hydrogen Peroxide) in Arabidopsis:
DAB staining method
• DAB (3,3'-diaminobenzidine) is oxidized by H2O2 : Leaves a dark brown precipitation.
• High colour intensity High precipitation High ROS levels
ROS levels in Arabidopsis
• WT: 1-6: Closely complemented line (WT-like)• Del1: 2-4: Not complemented line (cpr5-2-like)
Results/Conclusion
WT WT: 1-6 cpr5-2 Del1: 2-4
Overall experience/Things I learnt
• Plant techniques
• Molecular techniques
• Plant Physiology
• Literature
Acknowledgements
• Dr. Paul Dijkwel• Muhammad Faisal• Lab members (C5.19)• David, Rebecca, Satendra, Pam and Pam.
