维氏气单胞菌rpoN基因缺失株的构建及部分生物学特性分析

为了研究rpoN基因对维氏气单胞菌致病性的影响,采用同源重组的方法构建该基因缺失株。以该菌基因组DNA为模板,通过PCR扩增rpoN基因的上、下游同源臂;以上、下游同源臂胶回收产物为模板,P1-1/P2-2为引物进行重叠PCR扩增相应目的片段,从而获得rpoN上下游同源臂连接片段;将获取的连接片段连接pRE112,将连接产物转化至大肠杆菌DH5α感受态细胞,提取重组质粒pRE112-rpoN,经同源重组和抗生素筛选获得维氏气单胞菌rpoN基因缺失株ΔrpoN。同时,依然利用重叠PCR将目的基因片段与启动子相连接,而后与质粒p BBR-MCS相连接,最后将重组质粒pBBR-MCS-rpoN转化至缺失株ΔrpoN中,构建互补株C-rpoN。PCR鉴定和测序结果显示,成功构建出维氏气单胞菌rpoN基因缺失株ΔrpoN和互补株C-rpoN。生物学特性分析试验结果表明rpoN的缺失导致维氏气单胞菌TH0426菌落形态改变、生物被膜形成能力下降、动物致病性减弱,鞭毛断裂,运动性丧失。本研究中成功构建了维氏气单胞菌TH0426株rpoN基因的缺失株ΔrpoN,为进一步研究维氏气单胞菌减毒疫苗和rpoN基因的功能奠定了基础。

Construction and partial biological characteristics analysis of rpoN gene mutant in Aeromonas veronii

In order to study the pathogenic effect of rpo N gene on Aeromonas veronii,the gene deletion strain was constructed by homologous recombination.The upstream and downstream homologous arms of the rpo N gene were amplified by PCR using the genomic DNA of A.veronii TH0426 strain as a template.The upstream and downstream homologous arm gel recovery products were used as a template,and P1-1/P2-2 was used as a primer to carry out overlap PCR amplification of the corresponding target fragment,thereby obtaining a homologous arm ligation fragment upstream and downstream of rpo N gene.The obtained ligation fragment was ligated and cloned into p RE112 to obtain p RE112-rpo N.The ligation product was transformed into Escherichia coli DH5αcompetent cell,and the recombinant plasmid p RE112-rpo N was extracted.The A.veronii rpo N deletion strainΔrpo N was obtained by homologous recombination and antibiotic screening.Meanwhile,the target gene fragment was ligated to the promoter by overlap PCR,and then ligated to the plasmid p BBR-MCS.Finally,the recombinant plasmid p BBR-MCS-rpo N was transformed into the deletion strainΔrpo N to construct a complementary strain C-rpo N.PCR identification and sequencing results showed that the A.veronii rpo N deletion strainΔrpo N and the complementary strain C-rpo N were successfully constructed.The results of biological characteristics analysis showed that the loss of rpo N resulted in the morphological changes of colony of A.veronii TH0426,decreased biofilm formation ability,decreased adhesion and invasion ability,decreased pathogenicity of animals,decreased virulence,flagellar rupture and loss of motility.This study success-fully constructed theΔrpo N,a deletion strain of rpo N from A.veronii TH0426 strain,which laid the foundation for further study of attenuated vaccine and rpo N gene function of A.veronii.