【作者】 王铁霖；

【导师】 赵廷昌；

【作者基本信息】 中国农业科学院 ， 植物病理学， 2012， 硕士

【摘要】 瓜类果斑病是葫芦科作物上的一种种传病害，在世界多国和我国多个省份均有发生，给当地的西甜瓜种植业造成了严重的经济损失。其病原为西瓜嗜酸菌(Acidovorax citrulli,Schaad et al.2009)，革兰氏阴性菌。与大多数革兰氏阴性菌一样，该病原菌具有luxR/luxI群体感应系统，调节细菌相关生物学功能。本论文运用同源标记置换法，获得了瓜类果斑病菌(Acidovorax citrulli)群体感应信号系统功能相关基因3个突变菌株：△luxI单基因缺失突变株、△luxR单基因缺失突变株以及△luxR/luxI双基因缺失突变株。通过比较野生菌株和突变菌株间的生物学性状差异研究了luxR/luxI群体感应系统的功能。主要获得以下结论：(1)群体感应信号检测结果：与野生菌株相比，突变菌株△luxI和△luxR/luxI中检测不到信号物质的存在，△luxR菌株中仍有信号物质的蓝色显色反应。该实验结果进一步证明了luxI基因是自诱导物合成酶的基因，而luxR是合成信号接受载体的基因。(2)致病性测定结果显示：突变菌株与野生型相比，致病力明显减弱，说明群体感应系统的功能基因在细菌的致病力方面起着极其重要的作用。(3)生理生化测定结果：稳定期前，突变菌株与野生型菌株在KB培养基中生长速度一致，到稳定期，突变菌株生长能力稍微变弱；运动能力及胞外多糖的产生量与野生菌株没有明显差异。(4) Real-Time qPCR定量检测果斑病菌5个相关毒性基因及2个群体感应相关基因的表达情况。结果显示：与野生型相比，所选取的7个基因在突变菌株中的表达量均下调，说明群体感应系统对毒性基因的表达为正调控关系。并且，群体感应系统功能基因luxI在△luxR突变株中表达量降低；luxR基因在△luxI突变株中表达量降低。(5)萃取果斑病菌的群体感应信号物质，利用超高效液相色谱与质谱联用(UPLC-MS/MS)法鉴定，信号物质主要为N-(3-Oxo-octanoyl)-L-homoserine lactone（OOHL）；将该物质按照检测浓度与△luxI单基因缺失突变株混合接种，突变株的致病力恢复，实验结果表明群体感应信号物质与果斑病菌的致病性相关，与致病性测定实验结果一致。更多还原

【Abstract】 Bacterial fruit blotch of melons, caused by Acidovorax citrulli, is a seed borne disease that causesserious damages to the plants of cucurbits in worldwide. As most gram-negative bacteria, the pathogenhas luxR/luxI quorum sensing system to regulate its biological functions.Application the method of homologous recombination to construct three mutants related to the QSsignal systemic functional genes, containing luxR and luxI single gene deletion mutants and luxR/luxIdouble gene deletion mutant. luxR/luxI QS system function was studied by comparing the differnecebetween the wild type and mutant type strains. The results are as follows:(1) Results of AHLs detection：Compared with wide-type strain signal substances cannot bedetected in△luxI and△luxR/luxI because of the deletion of luxI, while△luxR can still display ablue chromogenic reaction due to the existence of luxI. This result is consistent with the conclusion thatluxI is the gene producing signal substances and luxR is the gene generating the carrier of the signalsubstances.(2) Results of pathogenicity determination：the mutants displayed the reduced in virulence, theresult showed that QS system played an important role in pathogenicity of Acidovorax citrulli.(3) Results of physiology and biochemistry bioasaay: the mutant strains are not attenuated ingrowth rate until stationary phase.There is no obvious difference in motility ability extracellularpolysaccharide secretion.(4) Results of Real-Time PCR：The expression of five genes related to virulence including luxI andluxR genes were detected quantitatively by Real-Time PCR, and the result revealed that the expressionof the seven genes we selected decreased in mutant strains, displayed negative regulation, similar withthe result of pathogenicity of the mutation-type strains. Results of Real-Time qPCR：The expression offive genes related to virulence and two gene including luxI and luxR related to quorum sensing werequantitatively detected by Real-Time qPCR. Results showed that the expression of the seven genesdecreased in mutant strains, displayed positive regulation. And the quorum sensing system related genesluxI was expressed at a lower level in△luxR and luxR was expressed at a lower level in△luxIrespectively.(5) The AHLs of bacterial fruit blotch of melons were extracted, and identified quantitatively byUPLC-MS/MS as OOHL. The pathogenicity of△luxI to melon seedlings was recovered after addingthe OOHL.更多还原