大白菜在接菌条件下cDNA文库的构建及应用初报

cDNA library of Chinese cabbage inoculated by Xanthomonas campestris has been constructed by using SMART (Switching mechanism at 5’ End of the RNA transcript) and recombination methods. The double strand cDNAs were cloned into pGADT7-Rec and introduced into yeast AH109 strain. The transformation rate was about 2.71×106 transformants per 1 µg pGADT7-Rec, and the recombinant rate would be above 86.4％ A bait system was constructed by cloning the avrXccE1, avirulence gene of Xanthomonas campestris, onto the plasmid pGBKT7 and being introduced into yeast Y187 strain. After the assays of self-activation of the bait system and its toxicity to the Y187 yeast cells, the hybridization between yeast strains AH109/cDNA and Y187/avrXccE1 was conducted by the preliminary screening on the medium containing SD/-Ade-His-Leu-Trp and the re-screening on the medium containing SD/-Ade-His-Leu-Trp-X-α-Gal for twice. The positive colonies were selected and the double strand cDNA harboured were sequenced. Four candidate target genes from Chinese cabbage were fished out from the cDNA library by using avrXccE1 as the bait. The results of BlastX of the cDNA sequenced to the GenBank database in NCBI indicated that the four candidate target genes might encode for ubiquitin-conjugating enzyme 11, thiol methyltransferase, MAP Kinase 18 and chitinase gene, respectively. The immunoprecipitation tests have been being conducted to confirm the natural interaction between the avirulence gene avrXccE1 from the pathogen Xanthomonas campestris and the candidate target genes from Chinese cabbage.