瓜类细菌性果斑病菌hrcN基因的生物学功能分析

Biological function analysis of hrcN gene of Acidovorax citrulli

以分离自西瓜上的Aac5菌株为例,通过同源重组的方法,构建了hrc N基因插入缺失突变体,通过PCR方法和Southern blot验证突变菌株,对突变体进行致病性、致敏性、生长曲线和运动性测定。为明确hrc N基因与其他基因的关系,通过实时荧光定量PCR法定量检测了hrp A、hrc V、hrc U、Lux I、LuxR 5个基因的表达量。结果显示:与野生型相比,突变体致病力和致敏性明显减弱,致病时间延迟,群体感应信号减弱,生长能力明显下降,运动性减弱,互补菌株只能恢复部分功能;5个基因在突变体中的表达量均上调,hrc N基因与这5个基因之间均为负调控关系。说明hrc N基因在果斑病菌致病能力上发挥重要作用。

Bacterial fruit blotch of melons caused by Acidovorax citruUi（ Schaad et al.2008） is a wodd-wide disease and gives rise to serious damages on the cucurbits plants. Strain Aac5 isolated from watermelon was a mutant constructed with an inner inserted deletion of hrcN gene by using homologous recombination. This mutant strain was confirmed by PCR and Southern blot. The mutant displayed reduces in virulence, hypersensitive reaction, quorum sensing, growth ability and motility compared to wild type. All these phenotype changes could be partially restored through complement mutant by introducting hrcN. In order to understand the regulation relationships between hrcN and other related genes, three genes （ hrpA, hrcV, hrcU） related to pathogenicity, two genes （LuxI, LuxR）related to quorum sensing and a housekeeping gene（pilT） were selected to determine the expression by Real-Time qPCR. The results showed that the expressions of five selected genes in mutant strains were increased compared to the wild type. Thus the relationship between hrcN and five selected genes displayed a negative regulation. All the results showed that hrcN gene played an important role in pathogenicity of Acidovorax citrulli.