溶藻弧菌Ⅲ型分泌系统vscC基因缺失株的构建

Construction of vscC in-frame Deletion Mutant of Vibrio alginolyticus

[目的]构建未引入任何遗传标记物的溶藻弧菌vscC基因框内缺失突变株。[方法]首先以溶藻弧菌ZJ51-O基因组DNA为模板,用重叠延伸PCR(gene splicing by overlap extension PCR,SOE-PCR)扩增法构建体外vscC基因缺失片段;扩增产物经酶切后连接至自杀质粒pDM4,再转化入大肠杆菌SY327进行克隆,筛选阳性克隆子并进行PCR分析鉴定;提取阳性克隆子的自杀重组质粒pDM4-△vscC,用热休克法将其转入到大肠杆菌S17-1中,再经接合生殖的方式将自杀重组质粒导入溶藻弧菌ZJ51-O菌株中,利用抗生素筛选策略与蔗糖反向筛选系统,挑选疑似目的突变菌株,最后利用PCR鉴定与测序分析进行确证。[结果]溶藻弧菌ZJ51-O的vscC基因框内缺失突变株构建成功。[结论]该研究为进一步研究vscC基因的功能和T3SS在溶藻弧菌致病过程中所起的作用奠定基础,同时也为溶藻弧菌的其他基因突变株的构建和研究提供了参考和试验经验。

[Objective] The purpose of this study was to construct a vscC in-frame deletion mutant of Vibrio alginolyticus with no antibiotic resistance marker.[Method] The first vscC mutant molecules in vitro were generated by SOE-PCR and then ligated to a suicide vector pDM4 to construct a suicide recombinant vector pDM4-△vscC.To clone and replicate the recombinant vector,it was transformed to E.coli SY327 strain,and then positive clones were selected and proved by PCR analysis.After that,the pDM4-△vscC DNA was extracted in large numbers and transformed to the E.coli S17-1 strain that acted as a donor in bacterial conjugation using the heat shock method.The recombinant E.coli S17-1 strains then transferred the pDM4-△vscC to V.alginolyticus ZJ51-O by conjugation method;transconjugants were screened and selected sequentially using antibiotic selection strategy and sucrose based counter-selection system to find the suspected mutants wanted.Finally the suspected mutants were identified by PCR and confirmed by sequencing analysis.[Result] ZJ51-O△vscC was successfully constructed.[Conclusion] This study laid a foundation for further research on the function of vscC gene and molecular mechanism of type III secretion system in V.alginolyticus.Simultaneously,by the effective method other unknown functional genes in V.alginolyticus genome would be researched.