水稻白叶枯病菌毒性基因的克隆和功能分析

 通过DNA重组,用载体质粒pBR322和水稻白叶枯病菌(Xanthomonas campestris pv.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。

CLONING AND FUNCTIONAL ANALYSIS OF VIRULENCE GENE(S) FROM XANTHOMONAS CAMPESTRIS PV.ORYZAE

The Tn5-containing BamHI fragment from Tn5-generated mutant XcoM 3105 was cloned in pBR322 to yield the plasmid pVIR1(11.6Kb) for probeing virulence gene(s) of Xanthomonas campestris pv.oryzae.A genomic library of Xco wildtype strain JXO Ⅲ was constructed in Escherichia coli using the broad host range cosmid vector pSa747.8 clones(pNAX3101-3108) corresponding wildtype DNA fragments were identified by colony hybridization and southern analysis from the library.When a cosmid clone pNAX3103 was conjugated in to XcoM3105 by triparent mating,the conjugation fraquences being 2.06×10^-7 conjugants per recipent cell,pathogenicity and amylase activity of XocM3105 were restored as same phenotype of wild type strain JXO Ⅲ.It was therefore suggested that the cloned fragmant contained the functional virulence gene(s) and there was a negative regulation between the virulence gene(s) and amylase gene(s).