【作者】 宋俊贤；

【导师】 胡白石；

【作者基本信息】 南京农业大学 ， 植物病理学， 2008， 硕士

【摘要】 由梨火疫病菌Erwinia amylovora引起的蔷薇科植物的火疫病是一类毁灭性的细菌病害,造成严重的经济危害。为了有效防治病害的发生及危害,人们对其致病因子的分子机理作了大量的研究。而许多植物病原细菌毒性因子的产生都依赖于群体感应(quorum sensing,QS)系统的调节。LuxS蛋白参与细菌群体感应信号分子autoinducer-2(AI-2)合成的最后一步。本研究在实验室已有的研究基础上构建了Erwinia amylovora strainNCPPB1665(Era NCPPB1665)的luxS突变体(Ea△luxS)并且对突变体的表型进行了初步的分析。研究发现Ea△luxS影响细菌的游动性、胞外多糖的分泌以及对双氧水的耐受能力。分别将Era NCPPB1665、Ea△luxS、功能互补菌株Ea△luxS(pPRO-luxS)和空载体对照Ea△luxS(pPROEXHta)接种梨树叶柄,发现接种突变体Ea△luxS和空载体对照Ea△luxS(pPROEXHta)的叶片发病情况比野生型和功能互补菌株Ea△luxS(pPRO-luxS)明显降低。这表明luxS基因影响Era NCPPB1665重要毒性基因的表达,因此,干扰luxS基因的表达可能能成为控制梨火疫病菌的一种潜在方式。为了进一步明确luxS基因是否影响Era NCPPB1665中hrp基因的表达,我们在E.amylovora Ea321的致病岛中挑选了八个hrp相关基因,分别为dspE、hrpW、hrpA、hrpB、hrcJ、hrpL、hrpJ、hrpY。同时,根据Sanger Institute(www.sanger.ac.uk)网站中公布的E.amylovora基因组序列,对八个相关基因分别设计引物。提取野生型Era.NCPPB1665、突变体Ea△luxS、功能互补菌株Ea△luxS(pPRO-luxS)和空载体对照Ea△luxS(pPROEXHta)的总RNA,利用反转录差异显示技术研究luxS基因对八个hrp相关基因表达的影响情况。研究发现,luxS基因并不影响hrp相关基因的表达。更多还原

【Abstract】 Fire blight,caused by Erwinia amlyvora,is one of the most devastating and difficult-to-control diseases.Till now,a number of researches were focused on the molecular mechanisms of the virulence in E.amlyvora for control of this disease. Interestingly,many virulence factors in different phytopathogenic bacteria are regulated by Quorum sensing(QS) system.Additionally,the autoinducer 2(AI-2) was identified in E.amylovora in our previous study.The enzyme LuxS catalyses the last step in the production of autoinducer-2 (AI-2).In this study,the luxS mutant of Era NCPPB1665(Ea△luxS) has been constructed and the phenotypes of defined luxS mutant have been characterized based on our laboratory research.Inactivation of luxS in Era NCPPB 1665 impaired motility, extracellular polysaccharide production and tolerance to hydrogen peroxide. Inoculated Era NCPPB1665,Ea△luxS,the complemented strains Ea△luxS (pPRO-luxS) and negative control Ea△luxS(pPROEXHta) respectively to pear petiole,we found lesion length caused by Ea△luxS and Ea△luxS(pPROEXHta) was shorter then Era NCPPB1665 and Ea△luxS(pPRO-luxS).It showed that luxS gene infects expression of some virulence genes in Era NCPPB1665 and interference of luxS might be a potential way for controlling fire blight.In order to confirm whether luxS gene affect the expression of the hrp gene in Era NCPPB1665,eight hrp related genes were chosen from E.amylovora Ea321 for further study.Meanwhile,total RNA of strain Era.NCPPB1665,Ea△luxS,Ea△luxS (pPRO-luxS) was isolated,respectively,and reverse transcription(RT)-PCR was performed to study the expression of hrp gene.Our results indicated that the mutation on luxS did not affect the expression of hrp related genes.更多还原