水稻白叶枯病菌tatB基因的克隆及功能分析

Cloning and functional characterization of tatB gene from Xanthomonas oryzae pv. oryzae

根据水稻白叶枯病菌(Xanthomonas oryzae pv.oryzae,Xoo)PXO99的tatB基因序列设计引物,采用PCR方法从PXO99中克隆了tatB基因,命名为tatBXoo。通过同源重组的方法,构建了tatBXoo的插入突变株TBM,在含有X-gluc的抗性平板上筛选,并经Southern blot杂交验证确认。表型测定结果表明:与野生型相比,tatB突变株在水稻(寄主)上的致病性减弱,但在烟草(非寄主)上仍具有引起过敏反应的能力;tatB突变导致Xoo致病相关的重要表型如胞外多糖减少,游动性及趋化性减弱。但是,tatB突变不影响Xoo在基本培养基(MMX)中正常生长、胞外纤维素酶产生和生物膜形成。

A tatB homologue,designated as tatBXoo,was cloned from the genomic DNA of Xanthomonas oryzae pv.oryzae（Xoo）strain PXO99 by PCR amplification.A tatBXoo disruption mutant was successfully generated by a single cross-over event,selected by resistance on NA medium with X-gluc,and confirmed by Southern blot analysis.Phenotypic tests showed that the tatB mutant exhibited a significant reduction of virulence on host rice,but had the wild-type ability in triggering hypersensitive response in non-host tobacco.Meanwhile,the mutation of tatB impaired Xoo′s three virulence-associated functions,including extracellular polysaccharide（EPS）production,cell motility and chemotaxis.However,the disruption of tatB did not impair Xoo′s growth ability in MMX medium,production of extracellular cellulase and biofilm formation.