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通过DNA重组,用载体质粒pBR322和水稻白叶枯病菌(Xanthomonas campestrispv.oryzae Dye)毒性基因突变体XcoM3105带有转座子Tn5的毒性基因片段,构建了11.6kb的探针质粒pVIRl。用α-32P-dATP标记该探针,从构建在粘粒pSa747上的病菌野生型菌株JXO Ⅲ染色体基因文库中,通过菌落原位杂交和Southern杂交,筛选了8个毒性基因的克隆(pNAX3101～3108。将其中一个毒性基因克隆pNAX3103通过三亲交配接合法转移到XcoM3105中,接合频率为2.06×10~(-7)。功能互补分析表明,pNAX3103可以互补突变体XcoM3105,恢复致病性,同时也能互补其淀粉酶(Amy)活性表型,由原来的淀粉酶阳性恢复为阴性反应。因此可以认为,被克隆的毒性基因片段具有完整的致病功能,毒性基因与淀粉酶基因之间可能存在着某种负调控关系。
A 11.6kb probe plasmid pVIR1 was constructed by DNA recombination using the virulence gene fragment of transposon Tn5 from vector plasmid pBR322 and Xanthomonas campestrispv. Oryzae Dye virulence gene mutant XcoM3105. The probe was labeled with α-32P-dATP. Eight virulence genes were cloned by in situ hybridization and Southern blotting from the wild type JXO Ⅲ gene library constructed on cosmid pSa747 (pNAX3101 ~ 3108. One of the virulence gene clones, pNAX3103, was transferred into XcoM3105 by mating mating method at a frequency of 2.06 × 10 ~ (-7) .Functional complementation analysis showed that pNAX3103 could complement XcoM3105 to restore pathogenicity, Also can complement its amylase (Amy) activity phenotype, from the original amylase positive returned to a negative reaction.Therefore, it can be considered, the cloned virulence gene fragment has a complete pathogenic function, the possibility of toxic genes and amylase genes There is some negative regulatory relationship.