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【目的】为了阐明可扩散性信号分子(diffusible signal factor,DSF)调控的鞭毛基因对水稻细菌性条斑病菌(Xanthomonas oryzae pv.oryzicola,Xoc)Rs105的致病性等方面的影响。【方法】采用PCR的方法克隆靶标基因flgDxoc和flgExoc,用同源重组法构建缺失突变体,测定突变体及其互补菌株的菌体形态、运动性、致病性及过敏性反应等表型,用反转录PCR(RT-PCR)的方法验证Rs105和ΔrpfFxoc(rpfFxoc基因的缺失突变体,不产生DSF)中flgDxoc、flgExoc表达量的差异。【结果】从Rs105基因组中克隆到flgDxoc、flgExoc基因,并证实这两个基因在基因组中均为单拷贝。PCR和Southern杂交结果显示,flgDxoc、flgExoc基因被成功敲除。与野生型相比,突变体的鞭毛产生能力丧失,游动性和趋化性能力减弱,接种水稻叶片显示其致病性部分减弱,基因互补可使其恢复。生长能力和对烟草叶片的致敏性无明显改变。RT-PCR结果显示,flgDxoc、flgExoc基因在ΔrpfFxoc中的转录水平明显降低。【结论】本试验表明:FlgD、FlgE是水稻细菌性条斑病菌鞭毛形成所必需的因子;进一步证明了DSF通过调控flgDxoc、flgExoc基因表达,从而影响条斑病菌的致病性等表型。为深入认识DSF对细菌性条斑病菌鞭毛基因簇的调控提供了科学依据。
【Objective】 The aim of this study was to elucidate the effects of diffusible signal factor (DSF) -mediated flagellar gene on the pathogenicity of Xanthomonas oryzae pv.oryzicola (Xoc) Rs105. 【Method】 The target genes flgDxoc and flgExoc were cloned by PCR, and the deletion mutants were constructed by homologous recombination. The phenotypes of the mutant and its complementary strains were determined, such as morphology, motility, pathogenicity and allergic reaction. Differences in the expression levels of flgDxoc and flgExoc in Rs105 and ΔrpfFxoc (deletion mutants of the rpfFxoc gene without generating DSF) were verified by reverse transcription PCR (RT-PCR). 【Result】 The flgDxoc and flgExoc genes were cloned from the Rs105 genome and confirmed that both genes were single copies in the genome. PCR and Southern hybridization results showed that flgDxoc, flgExoc gene was successfully knocked out. Compared with the wild type, the mutants lost the ability of producing flagella and the ability of mobility and chemotaxis weakened. Inoculation of rice leaves showed that the pathogenicity of the mutants was weakened and the complementation of genes could restore them. Growth ability and sensitization to tobacco leaves did not change significantly. RT-PCR results showed that flgDxoc, flgExoc gene transcription in ΔrpfFxoc significantly reduced. 【Conclusion】 The results showed that: FlgD and FlgE are necessary factors for the formation of flagella of bacterial leaf spot of Bacterial leaf blight. It is further proved that DSF can affect the pathogenic phenotype such as flgDxoc and flgExoc by regulating the expression of flgDxoc and flgExoc genes. This provided a scientific basis for further understanding of DSF regulation of flagella gene cluster of Bacterial leaf spot disease.