Cloning And Expression In Vivo The Resistant-Related Gene Of Arabidopsis Against Botrytis Cinerea

51,600 Arabidopsis T-DNA mutants and its wild type Columbia which being resistantto Botrytis cinerea inoculated by the pathogen, a Botrytis-susceptible mutant named⊿bosA,which is single copy of foreign DNA in the genome of the Arabidopsis thaliana was gotten.The left flanking sequence of T-DNA insert was obtained by TAIL-PCR, then analyzed bythe software of BLAST on the website http://www.arabidopsis.org andwww.ncbi.nih.gov, we were sure that the locus of the insert was in the fourth extron ofAT1g01800 between the bases of 956 and 957. BosA encodes a short-chaindehydrogenase/reductase (SDR) family protein which was expressed in E.coli strain BL21showed no direct resistance to B.cinerea. However, it really mediates responses to bothbiotic and abiotic stress agents.Among the51,600 Arabidopsis T-DNA mutants, 12 Botrytis-susceptible mutants weregotten. The further identification of histological and morphological research was carriedout, it's turn out to be that, the Botrytis-susceptible mutants, 1d after treaded mutants withBotrytis, appeared several dead cells, and with the time going, the quantity of dead cellswas increased, until 5d, eyeable symptom emerged. Then after 8d, the whole leaf was dead,while the Co1-0 only appeared little dead cells and no extended.Judging by the information given by the Southern Blotting analysis, we made theNo.134, which was named⊿bosA, as our material to further investigate. TAIL-PCR wasused to amplicated the flanking sequence of the known fragment, we had optimized thesysterm and the factors that affect the efficiency of TAIL-PCR. The left of the T-DNA wasobtained by TAIL-PCR, Analyzed by software of BLAST on the websitehttp://www.arabidopsis.org, and www.ncbi.nih.gov made us know that the locus of theinsert was between the bases of 956 and 957 of gene AT1g01800, named bosA. We can besure that it is related to the resistance.To better understand the mechanisms of BOSA function, we expressed the gene inE.coil strain BL21, extracted the active protein, and co-incubated it with Botrytis cinerea,there was no difference of the pathogen's growth and development before and afterinduced expression by IPTG, indicating that BOSA may not resist the Botrytis directly. Three days after inoculation with another necrotroph pathogen A. brassicicola,⊿bosAleaves showed significantly larger disease lesions than did A. brassicicola inoculatedwild-type leaves. While inoculated it by bacterial pathogen, Pst.DC3000,⊿bosA wasmore susceptible than the wlid-type. All of these indicated bosA play a role in the basicresistance rather than the gene-gene resistance. In addition, a series of abiotic stress on⊿bosA were carried out, compared with wild-type, it turned out to be that boxA mediatesresponses not only to biotic but also abiotic stress agents. The results of DAB staining indicatedthat, the enhanced susceptibility to Botrytis cinerea of⊿bosA positively correlated withthe levels of H₂O₂ produced.