Post on 21-Dec-2015
Plant ThermotolerancePlant Thermotolerance- - hot1-3 hot1-3 (HSP101)(HSP101)
HORT 301HORT 3019/30/20089/30/2008
Your goal:Discover New Genes that function for Thermotolerance
Choose one of the following:
Reverse genetic approach
Forward genetic approach
Reverse genetic approach for thermotolerancemutant plants are identified based on screening a large population harboring random mutations for a phenotypic change
1. Select a gene or genes - Complete genome sequences provide the information of genes or
gene family
Select tomato HSF1a
Baniwal et al., 2004 J. Biosci.
2. Generate mutants or overexpression lines 3. Test hypothesized responses (Thermotolerance)
ControlOver-expression
Co-suppression
Mishra et al., 2002 Genes and Development
Back
Forward genetic approach for thermotolerancemutant plants are identified based on screening a large population harboring random mutations for a phenotypic change
1. Select a biological process - Acquired thermotolerance for Dark-induced hypocotyl growth
24C 45C 2 hr 38C 1.5hr45C 2 hr
Chan Yul’s experiment
2. Generate a random and highly redundant mutant population Ethyl methanesulfonate – chemical modification of nucelotides C T
Forward genetic approach for thermotolerance
To achieve saturation of EMS mutagenesis in Arabidopsis, at least 125,000 M1 lines should be generated (Jander et al., 2003; Kim et al., 2006)
3. Screen a large number of mutagenized M2 plants 17,000 M2 seeds were screened (from estimated 2,100 M1 parents)
Sow 17,000 seeds on the medium in the square plates (As we did last week!)
Mark the seedlings that do not show acquired thermotolerance
Rescue the seedlings
Forward genetic approach for thermotolerance
Hong and Vierling, 2000 PNAS
5. Confirm by Complementation test
Hong and Vierling, 2000 PNAS
So, HSP101 gene functions for acquired thermotolerance
6. Confirm by another allele (hot1-3)
Hong and Vierling, 2000 PNAS
HSP101 gene (At1g74310)
hot1-3 is a T-DNA insertion mutant that eliminates HSP expression
HSP101HSP101
Function of HSP101: Molecular chaperone; ATP-dependent dissociation of protein aggregates
Baniwal et al., 2004 J. Biosci.