UCOL talk (031214)
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This document summarizes an internship presentation on work with the CPR5 gene in Arabidopsis thaliana. The presentation discusses: 1) Using Arabidopsis as a model organism and details about the CPR5 gene. 2) A project to uncouple the different processes CPR5 is involved in using a complementation strategy. 3) Results showing that complemented lines were successfully generated through PCR amplification and restriction enzyme digestion of the CPR5 gene.
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GenomicsPubudu Gokarella
油
Genomics is a sub-discipline of genetics focused on the mapping and analysis of genomes, differentiating from traditional genetics by examining entire systems rather than individual genes. The Human Genome Project played a critical role in sequencing the complex human genome, aiming to identify genes and their functions while addressing ethical concerns. The field of genomics has applications in medicine, agriculture, and understanding evolutionary processes, with extensive sequencing efforts ongoing for various organisms.Ad
UCOL talk (031214)
- 1. Internship presentation Sunmeet Singh Bhatia
- 2. 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)
- 3. 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)
- 4. 24-day air grown Early leaf senescence cpr5-2 plants WT cpr5-2 ROS generation HR execution Resistance: Bacterial & fungal Aberrant Leaf trichomes CPR5 mutant abnormalities cpr5-2WT 際際滷 Courtesy: Muhammad Faisal WT cpr5-2
- 5. Project Try to uncouple the different processes of Arabidopsis in which CPR5 is involved.
- 6. How do we do that? Complementation!
- 7. WT cpr5-2 COMPLEMENTATION STRATEGY Employed strategy for motifs/sites elucidation Constructs Wild type phenotype restorationRescue ?
- 8. Genotyping Genotyping the complemented lines to confirm the success of complementation.
- 9. Genotyping Genomic DNA Genotyping strategy PCR using gene specific primers Digestion of amplified product
- 10. 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 1.0kb 0.8kb 0.6kb 0.4kb 0.2kb 1.5kb 1.0kb 0.8kb 0.6kb 0.4kb 0.2kb
- 11. 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
- 12. 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 1.5kb 1.0kb 0.8kb 0.6kb 0.4kb 0.2kb 1.5kb 1.0kb 0.8kb 0.6kb 0.4kb 0.2kb
- 13. Results/Conclusions Amplification only from complemented lines and Migration of expected number and size of DNA fragments on gel confirms successful complementation
- 14. 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
- 15. Results/conclusions -40 -20 0 20 40 60 80 100 120 140 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) 1-branch 2-branch 3-branch 4-branch ~WT >WT Partially complemented lineNot complemented line
- 16. 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)
- 17. 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
- 18. 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
- 19. Overall experience/Things I learnt Plant techniques Molecular techniques Plant Physiology Literature
- 20. Acknowledgements Dr. Paul Dijkwel Muhammad Faisal Lab members (C5.19) David, Rebecca, Satendra, Pam and Pam.
Editor's Notes
- #4: https://www.arabidopsis.org/servlets/TairObject?id=135144&type=locus
- #5: OLD1 was identified from Arabidopsis during EMS screening and has been implicated in various integral processes. The first implication is HR execution. Actually when a pathogen attacks plant and is recognized by plant defense system then plant defense system immediately strive to subvert infection by a robust and local collapse of cells called (HR) hypersensitive response. As a result of programmed cell death, these kind of patches appear on leaves, which is a unique feature of programmed cell death. So mutants displaying such HR responses in the absence of pathogen were proven to be important and potential candidate in order to study PCD.
- #8: To achieve our goals, we adopted complementation strategy in which the defective/mutant gene is rescued by transforming and expressing WT gene. As we know, OLD1 complements all of its phenotype so identify crucial regions, the old1 mutant will be complemented with different constructs with different mutations, and will see how many of these manages to rescue either completely or partially.