本文選題：副溶血弧菌　切入點(diǎn)：H-NS　出處：《軍事醫(yī)學(xué)》2017年06期 　論文類(lèi)型：期刊論文

【摘要】：目的研究副溶血弧菌H-NS對(duì)vp1667的轉(zhuǎn)錄調(diào)控機(jī)制。方法提取副溶血弧菌hns突變株(Δhns)和野生株(WT)的總RNA,采用實(shí)時(shí)定量RT-PCR的方法驗(yàn)證H-NS對(duì)vp1667的轉(zhuǎn)錄調(diào)控關(guān)系;采用引物延伸實(shí)驗(yàn)研究vp1667的轉(zhuǎn)錄起始位點(diǎn),并根據(jù)產(chǎn)物的豐度判斷H-NS對(duì)vp1667的調(diào)控關(guān)系;將vp1667的啟動(dòng)子區(qū)克隆入p HRP309質(zhì)粒的β-半乳糖苷酶基因上游,構(gòu)建Lac Z重組質(zhì)粒,并將該重組質(zhì)粒轉(zhuǎn)入Δhns和WT中獲得Lac Z菌株。通過(guò)Lac Z報(bào)告基因融合實(shí)驗(yàn)研究H-NS對(duì)vp1667的調(diào)控關(guān)系。PCR擴(kuò)增vp1667的啟動(dòng)子區(qū)序列并純化His-H-NS蛋白,通過(guò)凝膠阻滯實(shí)驗(yàn)(EMSA)研究His-H-NS對(duì)vp1667啟動(dòng)子區(qū)是否具有直接的結(jié)合作用;采用DNaseⅠ足跡實(shí)驗(yàn)研究HisH-NS對(duì)vp1667啟動(dòng)子區(qū)的具體結(jié)合位點(diǎn)。結(jié)果與結(jié)論引物延伸結(jié)果顯示,vp1667只有一個(gè)轉(zhuǎn)錄起始位點(diǎn)T(-28)(翻譯起始位點(diǎn)為+1),且其轉(zhuǎn)錄活性受H-NS的抑制;EMSA和DNaseⅠ足跡實(shí)驗(yàn)結(jié)果顯示,His-H-NS不能結(jié)合到vp1667的啟動(dòng)子區(qū),表明H-NS只能間接抑制vp1667的轉(zhuǎn)錄。
[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，

本文編號(hào)：1638094