摘要
目的利用分子生物学实验研究OxyR对katY的转录调控机制。方法 PCR扩增katY的整个启动子区DNA序列全长,并纯化鼠疫菌His-OxyR蛋白,通过体外的凝胶阻滞实验(EMSA)和DNase I足迹实验研究OxyR对katY启动子区的结合位点;提取鼠疫菌野生株(WT)和oxyR突变株(ΔoxyR)的总RNA,采用引物延伸实验研究katY的转录起始位点,并根据产物的丰度判断OxyR对katY的调控关系;进一步采用实时定量RT-PCR的方法验证OxyR对katY的调控关系。结果体外实验结果表明OxyR能结合到katY启动子区-101到-48之间的碱基上;鼠疫菌katY有一个转录起始位点G(-26)(翻译起始位点为+1),其转录表达受OxyR的激活。结论 OxyR能直接结合到katY启动子区而激活其转录表达。
The aim of this study is to investigate the transcriptional regulation mechanism of leatY by OxyR in Yersinia pestis. The entire promoter-proximal region of the katY gene was amplified by PCR from Y. pestis strain 201. And the over expressed His-OxyR was purified under native conditions with nickel loaded HiTrap Chelating Sepharose columns （Amer sham）. Then, the electrophoretic mobility shift assay （EMSA） and DNase I footprinting experiment were applied to analyze the binding site of His OxyR to leatY promoter region in vitro. Total RNAs were extracted from the wide-type （WT） strain and the o.ryR null mutant （Ao：ryR）. Primer extension assay was employed to detect the promoter activity （the amount of prim er extension products） of leatY in WT and that in z^oxyR. And then, quantitative RT PCR was carried out to calculate the tran scriptional variation of leatY between the WT and Aoa＇yR. The in vitro experiments showed that His-OxyR bound to a single region from 101 bp to 48 bp upstream of katY. The primer extension assay detected only one transcriptional start site located al 26 bp upstream of katY, whose transcript was under positive control of OxyR. Our data suggest that the transcription of katY is directly activated by OxyR in Yersinia pestis.
出处
《中国人兽共患病学报》
CAS
CSCD
北大核心
2013年第9期850-853,861,共5页
Chinese Journal of Zoonoses
基金
病原微生物生物安全国家重点实验室开放研究基金资助项目(No.SKLPBS1112)
江苏省高校自然科学研究项目(No.12KJD310001)
江苏大学高级专业人才科研启动基金项目(No.10JDG044)~~
