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Establishment of pcr-based site-selected mutagenesis in 'arabidopsis' to inactive genes putatively involved in disease resistance

Objective



Research objectives and content
The DNA sequence of the Arabidopsis genome is being determined. The function of genes identified by sequencing will be a major focus of biological research. I will (with others) develop a 'mutation machine' in Arabidopsis which will facilitate the isolation of insertion mutations in any known DNA sequence. Analysis of mutant phenotypes will give information on gene function. The approach involves saturation insertion mutagenesis; identification of mutations in specific genes can then be achieved with a PCR screen. We will use the En(Spm)/l(dSpm) transposon system which is active in Arabidopsis. By selecting directly for transposed dSpm elements, we aim to produce at least 57,600 transposants. With this level of saturation, it should be possible to identify an insertion in any Arabidospsis gene (average size of 3.2 kb) with a probability of 82%. The mutation machine is being developed in the Jones lab to test whether certain genes whose induction or activation has been correlated with defense response really play an indispensable role in resistance. In particular, to test the mutation machine, I will target the following genes: (1) NADPH oxidase genes homologues from mammalian NADPH oxidases (2) calcium-dependent protein kinases (3) glucanases and chitinases. Training content (objective, benefit and expected impact)
Working on this project is very interesting to me for several reasons: -It will allow me to learn about plant pathology in an environment that is perfectly suited for this purpose -It will give me experience with the model plant Arabidopsis which I plan to continue using after this position -Finally, in addition of mutations that will be analyzed in this work, the mutation machine will provide material that will be useful for future studies. All three aspects will useful to me later as I plan to continue investigating the mechanisms involved in disease resistance. Links with industry / industrial relevance (22)

Funding Scheme

RGI - Research grants (individual fellowships)

Coordinator

THE SAINSBURY LABORATORY
Address
Norwich Research Park, Colney Lane
NR47UH Norwich
United Kingdom

Participants (1)

Not available
France