Isolation of genes involved in stress tolerance by activation tagging

Abstract

By the activation T-DNA tagging, we isolated a variety of Arabidopsis mutant lines with their abilities to grow under several stress conditions. The in planta transformation method was used to generate approx. 20,000 seeds of transgenic plants with pPCVIEn4HPT vector. Wild-type plants could not grow on the MS medium containing 0.6 µM paraquat under illumination at 100 µmol/m2/s. Among approx. 10,000 transformants, 102 dominant mutants could grow on the same medium. These paraquat resistant mutants (pqr) were analyzed by PCR using pPCVIEn4HPT-specific primers to clarify the existence of the T-DNA in the genome DNA. To determine whether the mutant phenotypes were caused by overexpression of adjacent genes, we characterized the T-DNA insertions in the several pqr lines. Plant genome adjacent to the T-DNA insertions were recovered by plasmid rescue or LA-PCR, and then analyzed by DNA sequencing and comparison with the Arabidopsis genome DNA in KAOS. In the pqr-61, 67, 74, 129, and 130, we identified the genes homogenous to thylakoid ascorbate peroxidase, cyt P-450, transport protein, kinesin-like protein, thyloid receptor interactor, Ser/Thr protein kinase, pactate lyase precursor, and aldose 1-epimerase and several unknown genes. Now we are determining the genes involved in the paraquat resistance in the respective pqr mutants by the micro array or the Northern blot analysis. On the other hand, wild-type plants exhibited chlorosis under low temperature and high light condition (2°C, 1000 µmol/m2/s). Among approx. 300 transformants, 8 dominant mutants did not show the chlorosis under the same stress condition.