【作者】 殷芳群；

【导师】 胡白石；

【作者基本信息】 南京农业大学 ， 植物病理学， 2011， 硕士

【摘要】 水稻黄单胞菌包含两个致病变种Xanthomonas orzae pv.oryzae(Xoo)和X.opvzae pv.oryzicola肠(Xoc),在水稻上分别引起白叶枯病和细菌性条斑病。为了阐明可扩散性信号分子(diffusible signal factor,DSF)调控的两个鞭毛基因flgD和flgE以及天冬酰胺合成酶基因(asparagine synthetase B,asnB)对水稻细菌性条斑病菌Rs105在致病性等方面的影响。在本研究中,我们克隆了flgD、flgE和asnB基因,并对其进行序列分析。用同源重组的方法构建相应基因的缺失突变株,明确了这三个基因对水稻细菌性条斑病菌在运动性,致病性和生长能力等方面的影响,以此来推知基因的功能；并进一步证明了DSF通过调控flgD、flgE和asnB基因得表达,从而影响条斑病菌的致病性等表型。为深入认识DSF对细菌性条斑病菌相关基因的调控提供了科学依据。(一)采用PCR的方法从Rs105基因组中克隆到flgD和flgE基因,并证实这两个基因在基因组中均为单拷贝。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ΔrpF(rpfF基因的缺失突变体,不产生DSF)中flgD、flgE表达量的差异。PCR和Southern杂交结果显示,flgD、flgE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,flgD、flgE基因在ΔrpfF中的转录水平明显降低。这表明：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flgD、flgE基因表达,从而影响条斑病菌的致病性等表型。(二)从Rs105基因组中克隆到asnB基因,并证实这个基因在基因组中为单拷贝。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、致病性和过敏性反应、在水稻叶片中的繁殖能力以及在添加不同氮源的MMX基本培养基里的生长能力等表型,用反转录PCR(RT-PCR)的方法验证Rs105和ΔrpfF(rpfF基因的缺失突变体,不产生DSF)中asnB表达量的差异。PCR和Southern杂交结果显示,asnB基因被成功敲除。与野生型相比,突变体接种水稻叶片显示其致病性基本丧失,基因互补可使其恢复。asnB基因的缺失严重影响了细菌性条斑病菌的生长,添加外源天冬酰胺可使其生长情况恢复。鞭毛产生能力,游动性和趋化性能力,降解胞外蛋白酶、纤维素酶的能力以及对烟草叶片的致敏性等无明显改变。RT-PCR结果显示,asnB基因在ΔrpfF中的转录水平明显降低。试验表明：asnB基因是细菌性条斑病菌正常生长所必需的,其突变可使细菌丧失致病性；进一步证明了DSF通过调控asnB基因表达,从而影响条斑病菌的致病性等表型。更多还原

【Abstract】 Xanthomonas oryzae pv. oryzae (Xoo) and X. oryzae pv. oryzicola(Xoc) cause leaf blight and leaf streak on rice respectively. To investigate functions of flagellar genes flgD and flgE and asparagine synthetase B gene (asnB) regulated by diffusible signal factor (DSF) in Xoc strain Rs105.In this study, the flgD, flgE and asnB genes were cloned and sequence analyzed from Xoc. We constructed the deletion mutants from Rs105 by using double crossover method, characterized the contribution of these genes to motility, virulence and bacterial growth to speculat the function of the genes. In addition, our results provided molecular evidences that the contribution of DSF-type quorum sensing to pathogen’s virulence might be.(Ⅰ) The flgD and flgE genes were amplified by PCR. We constructedΔflgD andΔflgE, the deletion mutants from Rs105 by using double crossover method, and determined cell morphology, motility, pathogenicity in host rice and hypersensitive response (HR) in nonhost tobacco. We tested the differential expression of flgD and flgE gene by reverse transcriptional polymerase chain reaction (RT-PCR) between the wide type andΔrpfF (the deletion mutant of rpfF gene, which could not produce DSF). PCR and Southern blot analysis demonstrated that the flgD and flgE genes were knocked out successfully. Both mutants were non-flagellated and significantly attenuated motility on the 0.3% semi-solid medium. The pathogenicity on rice were obviously attenuated inΔflgD andΔflgE compared to the wild type. All the changes in mutant could be restored through the complementation. However, there was no significant difference in bacterial growth in MMX medium and induction of HR between mutant (ΔflgD orΔflgE) and the wild type. In addition, the results of RT-PCR demonstrated that the transcription level of flgD and flgE were down-regulated inΔrpfF. This study showed that expressions of flgD and flgE were positively regulated by DSF, and necessary for flagellar hook assembly and flagellar structure in Xoc. Meanwhile, FlgD and FlgE contributed to pathogen’s virulence, motility and chemotaxis, but no deferences at growth rate in MMX medium and HR in nonhost. (Ⅱ) The asnB gene was amplified by PCR. We constructedΔasnB, the deletion mutants by using double crossover method, and determined cell morphology, motility, pathogenicity in host rice and hypersensitive response (HR) in nonhost tobacco, bacterial growth in rice and MMX medium with different nitrogen sources (Peptone, Yeast extract, Asparagine, Aspartic acid, Glutamine, Glutamate, Ammonium sulfate, Urea, Potassium nitrate or Alanine). We tested the differential expression of asnB gene by reverse transcriptional polymerase chain reaction (RT-PCR) between the wide type andΔrpfF. PCR and Southern blot analysis demonstrated that the asnB gene were knocked out successfully. Compaired with the wide type strain,ΔasnB was no pathogenicity on rice, the change in mutant could be restored through the complementation. The deletion of asnB gene had seriously affected the bacrerial growth, the change could be restored by adding exogenous asparagine. However, there was no significant difference in flagella production, motility and induction of HR between mutant and the wild type. In addition, the results of RT-PCR demonstrated that the transcription level of asnB was down-regulated in ArpfF. This study showed that expressions of asnB were positively regulated by DSF, and necessary for bacterial growth in Xoc. Meanwhile, AsnB contributed to pathogen’s virulence in nonhost rice.更多还原