拟核结合蛋白H-NS调控副溶血弧菌vp1667的转录

发布时间：2018-03-20 07:34

  本文选题：副溶血弧菌　切入点：H-NS　出处：《军事医学》2017年06期 　论文类型：期刊论文

【摘要】：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。
[Abstract]:Objective to study the transcriptional regulation mechanism of vibrio parahaemolyticus H-NS on vp1667. Methods Total RNAs of hns mutant (螖 hns) and wild strain (WTT) of Vibrio parahaemolyticus were extracted, and real-time quantitative RT-PCR was used to verify the transcriptional regulation of H-NS on vp1667. The transcriptional initiation site of vp1667 was studied by primer extension experiment, and the regulation of H-NS on vp1667 was evaluated according to the abundance of the product. The promoter region of vp1667 was cloned into the upstream of 尾 -galactosidase gene of p HRP309 plasmid, and the recombinant plasmid Lac Z was constructed. The recombinant plasmid was transferred into 螖 hns and WT to obtain Lac Z strain. The relationship between H-NS and vp1667 was studied by Lac Z reporter gene fusion. The promoter region sequence of vp1667 was amplified and the His-H-NS protein was purified. The direct binding of His-H-NS to the promoter region of vp1667 was studied by gel retardation assay (EMSA). DNase 鈪，

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