植物青枯菌Ⅵ型分泌系统核心基因vasK突变株的构建及其致病性的测定  

Construction and pathogenicity tests of the mutated strain of the core gene vasK of type Ⅵ secretion system in Ralstonia solanacearum

作　　者：张丽勍[1] 许景升[1] 徐进[1] 冯洁[1] 

机构地区：[1]中国农业科学院植物保护研究所,植物病虫害生物学国家重点实验室,北京100193

出　　处：《植物保护》2011年第4期33-37,共5页Plant Protection

基　　金：国家重点基础研究发展计划(“973”)项目(2009CB119200);国家科技支撑计划项目(2006BAD08A14);国家高技术研究与发展计划(“863”)项目(2006AA10Z432)

摘　　要：[目的]通过测试vasK基因突变株对番茄的致病力变化,评价该基因在青枯菌致病过程中的作用。[方法]根据青枯菌(Ralstonia solanacearum)中存在的Ⅵ型分泌系统基因簇中的核心基因vasK序列设计PCR引物,扩增并克隆vasK基因,将庆大霉素抗性基因(Gm)插入vasK基因内部,克隆至自杀质粒pK18mobsacB中,获得重组自杀质粒pK18-vasK-Gm。将自杀质粒电转化至青枯菌GMI1000感受态细胞中,采用同源重组双交换法,将野生型vasK基因置换。对vasK基因突变菌株进行三步筛选和PCR扩增鉴定。[结果]筛选获得了具有庆大霉素抗性的目标基因被抗性基因替换的青枯菌突变株(GMI1000-m)。土壤接种番茄青枯菌结果显示,突变株GMI1000-m的致病性较野生型GMI1000明显下降。[结论]vasK基因在青枯菌致病过程中具有重要作用。[Objective] The change in pathogenicity of the vasK mutant to the tomato was investigated to estimate the function of the vasK gene in the pathogenicity process of Ralstonia solanacearum.[Method] According to the sequence of vasK gene, a key component of type Ⅵ secretion system in Ralstonia solanacearum, PCR primers were designed to amplify vasK gene. vasK gene was mutated by inserting a gentamicin＆#8226;3acetyltransferase gene （Gm gene）, and the mutated vasK was inserted into suicide vector pK18mobsacB, resulting in pK18vasKGm. The vector pK18vasKGm was then introduced into R. solanacearum GMI1000. The vasK mutant, named GMI1000m, was generated by homologous recombination and selected by a threestep method. GMI1000m was identified by PCR amplification.[Result] The pathogenicity test of the mutant GMI1000m was decreased compared with the wild type GMI1000. [Conclusion] The vasK gene was an important factor in the pathogenesis of R. solanacearum.

关 键 词：青枯菌 Ⅵ型分泌系统 vasK基因 

分 类 号：Q753[生物学—分子生物学]