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目的研究副溶血弧菌H-NS对vp1667的转录调控机制。方法提取副溶血弧菌hns突变株(Δhns)和野生株(WT)的总RNA,采用实时定量RT-PCR的方法验证H-NS对vp1667的转录调控关系;采用引物延伸实验研究vp1667的转录起始位点,并根据产物的丰度判断H-NS对vp1667的调控关系;将vp1667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,并将该重组质粒转入Δhns和WT中获得Lac Z菌株。通过Lac Z报告基因融合实验研究H-NS对vp1667的调控关系。PCR扩增vp1667的启动子区序列并纯化His-H-NS蛋白,通过凝胶阻滞实验(EMSA)研究His-H-NS对vp1667启动子区是否具有直接的结合作用;采用DNaseⅠ足迹实验研究HisH-NS对vp1667启动子区的具体结合位点。结果与结论引物延伸结果显示,vp1667只有一个转录起始位点T(-28)(翻译起始位点为+1),且其转录活性受H-NS的抑制;EMSA和DNaseⅠ足迹实验结果显示,His-H-NS不能结合到vp1667的启动子区,表明H-NS只能间接抑制vp1667的转录。
Objective To study the transcriptional regulation of vp1667 by Vibrio parahaemolyticus H-NS. Methods The total RNA of Vibrio parahaemolyticus hns mutant (Δhns) and wild-type strain (WT) was extracted and the transcriptional regulation of Hp-p1667 was verified by real-time quantitative RT-PCR. The promoter region of vp1667 was cloned into the upstream of β-galactosidase gene of p HRP309 plasmid to construct Lac Z recombinant plasmid, Recombinant plasmids were transformed into Δhns and WT to obtain Lac Z strain. The Lac-reporter gene fusion assay was used to investigate the regulatory effect of H-NS on vp1667. The promoter region of vp1667 was amplified by PCR and the His-H-NS protein was purified. Whether His-H-NS had a direct binding effect on vp1667 promoter region was determined by gel-blocking assay (EMSA) Specific binding sites of HisH-NS to the vp1667 promoter region. RESULTS AND CONCLUSION: Primer extension results showed that vp1667 had only one transcription initiation site T (-28) (translation initiation site was +1) and its transcriptional activity was inhibited by H-NS. The results of EMSA and DNase I footprinting , His-H-NS can not bind to the promoter region of vp1667, indicating that H-NS can only indirectly inhibit the transcription of vp1667.