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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。
Xanthomonas oryzae pv. Oryzae (Xoo) and Xanthomonas oryzae pv. Oryzicola (Xoc) are the pathogenic bacteria in rice and cause bacterial blight (BB) and bacterial Bacterial leaf streak (BLS). In order to achieve the target gene mutation accurately and efficiently, we constructed a set of mutated system which is suitable for the site-directed insertion of Xoo and Xoc genes by using the pK18mobGⅡ vector. Through the homologous recombination between the homologous fragment and the target gene, the hrcV and hrpF mutants of Xoo and Xoc were successfully obtained. PCR and Southern blotting confirmed that pK18mobGII carries different size homologous fragments and can be integrated into specific sites of hrcV and hrpF. The homologous fragment of 200-400 bp can obtain the best mutation efficiency. The conversion efficiency of amphipathic junction is Conversion of 5 to 100 times. Toxicity test results showed that hrcV gene determines the pathogenicity of Xoo in rice. The establishment of pathogenicity-related gene insertion mutagenesis system laid the foundation of genetics research for studying the function of pathogenicity-related genes in Xanthomonas oryzae-rice interaction.