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目的构建副溶血弧菌calR的基因突变株和回补株,为研究CalR的功能奠定基础。方法 PCR扩增calR基因的同源臂融合片段,并直接克隆入自杀质粒p DS132中。通过接合转移的方式将重组自杀质粒转入副溶血弧菌RIMD2210633株(WT)中,利用同源重组的方法替换原始calR基因以构建calR无痕突变株(ΔcalR)。PCR扩增calR的基因序列,并将其直接克隆入p BAD33质粒中,构建回补质粒。将回补质粒转入到ΔcalR中,即得回补株(ΔcalR/calR∷p BAD33)。将vop N的启动子区克隆入p HRP309质粒的β-半乳糖苷酶基因上游,构建Lac Z重组质粒,并将该重组质粒分别转入WT/p BAD33、ΔcalR/p BAD33和ΔcalR/calR∷p BAD33中,通过测定并比较3株菌中β-半乳糖苷酶活性的差异来判定CalR对vop N的调控关系。结果与结论 Lac Z结果表明,CalR负调控vop N的转录,且回补株的回补效果良好,表明成功构建了副溶血弧菌calR基因的突变株和回补株,为后续对CalR的功能研究打下了基础。
OBJECTIVE: To construct the gene mutant and repair strains of calV of Vibrio parahaemolyticus, which laid the foundation for the study on the function of CalR. Methods The homology arm fusion fragment of calR gene was amplified by PCR and directly cloned into suicide plasmid p DS132. Recombinant suicide plasmids were transformed into Vibrio parahaemolyticus strain RIMD2210633 (WT) by conjugation and transfer. The original calR gene was replaced by homologous recombination to construct the calR mutant (ΔcalR). The gene sequence of calR was amplified by PCR and cloned directly into pBAD33 plasmid to construct the repl plasmid. Transfer the recombination plasmids into ΔcalR to recover the strain (ΔcalR / calR :: p BAD33). The promoter region of vop N was cloned into the upstream of β-galactosidase gene of p HRP309 plasmid to construct Lac Z recombinant plasmid and the recombinant plasmids were transformed into WT / p BAD33, ΔcalR / p BAD33 and ΔcalR / calR :: p BAD33, the regulation of Calo on vop N was determined by measuring and comparing the differences in β-galactosidase activity in the three strains. RESULTS AND CONCLUSION LacZ results showed that CalR negatively regulated the transcription of vop N, and the replenishing strains were well replenished. It showed that the mutant and complement of the calR gene of V. parahaemolyticus were constructed successfully, Research laid the foundation.