水稻黄单胞菌致病相关基因插入突变体系的建立

 水稻白叶枯病菌（Xanthomonas oryzae pv. oryzae, Xoo）和条斑病菌（Xanthomonas oryzae pv. oryzicola, Xoc）是水稻上的模式病原菌，分别引起水稻白叶枯病（bacterial blight, BB）和细菌性条斑病（bacterial leaf streak, BLS）。为了精确和高效地实现目的基因的突变，本研究利用pK18mobGⅡ载体，建立了一套适于Xoo和Xoc目的基因定点插入的突变体系。通过同源片段与目的基因间的同源重组，成功获得了Xoo和Xoc的hrcV和hrpF突变体。PCR和Southern杂交证实：pK18mobGⅡ携带不同大小的同源片段，能够整合于hrcV和hrpF的特定位点；200～400 bp的同源片段能够获得最佳的突变效率；两亲接合的转化效率是电转化的5～100倍。毒性测定结果显示，hrcV基因决定着Xoo在水稻上的致病性。致病相关基因插入突变体系的建立为研究水稻黄单胞菌与水稻互作中致病相关基因的功能奠定了遗传学研究基础。

Insertion mutagenesis in pathogenicity-associated genes of Xanthomonas oryzae

Two pathovars,Xanthomonas oryzae pv.oryzae(Xoo) and X.oryzae pv.oryzicola(Xoc),cause bacterial blight(BB) and bacterial leaf streak(BLS) of rice(Oryza sativa),respectively.In order to realize mutation in interesting genes of two pathovars accurately and efficiently,an insertion mutagenesis system was successfully established here with pK18mobGⅡ vector.The hrcV and hrpF mutants both of Xoo and Xoc were generated by allelic exchange between homologous fragments of the hrcV and hrpF genes,respectively.The pK18mobGⅡ,harboring different homologous fragments of hrcV or hrpF,was integrated into hrcV and hrpF gene loci.These events were verified by PCR amplification and Southern blot.200-400 bp homologous fragments for targeted genes by pK18mobGⅡ would lead to the higher mutation efficiency.Moreover,transformation efficiency of the recombined DNA plasmids to the recipient strain by conjugation was five to hundred times of that by electroporation.Virulence assay demonstrated that the insertion mutagenesis in hrcV gene led the complete loss of pathogenecity in host rice.Therefore,this mutagenesis system would facilitate the understanding of pathogenicity-associated genes when Xanthomonas oryzae interact with rice.