VPA0961对副溶血弧菌运动能力的调控

Regulation of swimming and swarming by VPA0961 in Vibrio parahaemolyticus

目的:构建vpa0961基因突变株及其回补株,并初步探究VPA0961对副溶血弧菌运动能力的调控作用。方法:以副溶血弧菌RIMD2210633野生株的基因组DNA为模板,PCR扩增得到vpa0961的同源臂融合片段,并克隆至自杀质粒pDS132多克隆位点。将pDS132重组质粒通过结合转移的方式转入野生株,进而通过同源重组方法替换原始vpa0961,制备vpa0961无痕突变株(Δvpa0961)。PCR扩增vpa0961整个编码区序列,并将其直接克隆入pBAD33质粒,获得重组回补质粒。将回补质粒结合转移到Δvpa0961中,然后通过特异性引物PCR鉴定并外送测序以获得回补株。将副溶血弧菌的预培养物接种于游动(swimming)和爬动(swarming)平板,37℃静置培养,比较不同菌株间菌苔直径的差异。采用qRT-PCR分析鞭毛蛋白基因lafA、flaB和flaF的mRNA表达水平。结果:特异性扩增及序列分析显示,成功构建vpa0961基因突变株和回补株;与野生株相比,Δvpa0961游动能力和爬动能力明显减弱(t=14.06,36.37,P<0.05);与野生株相比,Δvpa0961 lafA、flaB和flaF mRNA转录水平明显降低(t=185.2,56.36,50.24,P<0.05)。结论:成功构建vpa0961的基因突变株和回补株,且VPA0961对鞭毛基因的转录具有正调控作用。

Objective:To construct vpa0961 mutant strain and its complementary strain,and to investigate the regulation of VPA0961 on the motility of Vibrio parahaemolyticus.Methods:The homologous flanking fusion fragment of vpa0961 was amplified from Vibrio parahaemolyticus RIMD2210633,and then cloned into the suicide plasmid pDS132.Thereafter,the recombinant plasmid was transferred into Vibrio parahaemolyticus RIMD2210633 by means of binding transfer,and the original vpa0961 was replaced by homologous recombination to obtain vpa0961 mutant strain(Δvpa0961).The entire coding region of vpa0961 was amplified by PCR,and then cloned into pBAD33.The recombinant pBAD33 was transferred intoΔvpa0961 to construct the complementory mutant ofΔvpa0961.The preculture of Vibrio parahaemolyticus was inoculated on a swimming and swarming plate,and cultured at 37℃to compare the difference in diameter of the lawn between different strains.The mRNA levels of the flagellin genes lafA,flaB and flaF were detected by qRT-PCR.Results:The mutant and replenishing strains of vpa0961 were obtained.Compared with the wild-type strain,the swimming ability and swarming ability ofΔvpa0961 were significantly decreased(t=14.06,36.37,P<0.05);the mRNA levels of the lateral flagellin gene lafA and the polar flagellin gene flaB and flaF mRNA were significantly decreased(t=185.2,56.36,50.24,P<0.05).Conclusion:The deletion mutant of vpa0961 and its complementary strain were successfully constructed,and VPA0961 has a positive regulatory effect on the transcription of motility-related flagellar genes.