摘要
【目的】利用分子生物学实验研究鼠疫菌调控子OxyR对dps的转录调控机制。【方法】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dps的转录起始位点,并根据产物的丰度判断OxyR对dps的调控关系。进一步采用实时定量RT-PCR的方法验证OxyR对dps的调控关系。PCR扩增dps的整个启动子区DNA序列,并纯化His-OxyR蛋白,通过凝胶阻滞实验(EMSA)验证OxyR对dps启动子区是否具有直接的相互作用。利用大肠杆菌OxyR识别基序,预测鼠疫菌OxyR对dps启动子区的结合位点,从而得出鼠疫菌OxyR对dps的转录调控机制。【结果】鼠疫菌dps有一个转录起始位点G(-40)(翻译起始位点为+1),其转录表达受OxyR的激活;体外实验及生物信息学预测结果表明OxyR能结合到dps启动子区-111到-78之间的碱基上。【结论】OxyR能直接结合到dps启动子区而激活其转录表达。
[ Objective] To study the transcriptional regulation mechanism of dps by OxyR in Yersinia pestis. [ Methods ] Total RNAs were extracted from the wide-type （WT） strain and the oxyR null mutant （AoxyR）. Primer extension assay was used to detect the promoter activity （the amount of primer extension product） of dps in WT and that in z^oxyR. Then, quantitative RT-PCR was done to calculate the transcriptional variation of dps between the WT and AoxyR. The entire promoter-proximal region of the dps gene was amplified by PCR from Y. pestis strain 201. Over-expressed His-OxyR was purified under native conditions with nickel loaded HiTrap Chelating Sepharose columns （Amersham）. Then, the electrophoretic mobility shift assay （EMSA） was applied to analyze the DNA-binding activity of His-OxyR to dps promoter region in vitro. Finally, Escherichia coli OxyR consensus was deployed to predict the OxyR binding site to dps promoter region. [ Results] The primer extension assay detected only one transcriptional start site located at 40bp uptream of rips, whose transcript was under positive regulation by OxyR. The bioinformatics analysis indicated that His-OxyR bound to a single region from 11 l bp to 78bp upstream of dps. [ Conclusion] The transcription of dps was directly activated by OxyR in Yersinia pestis.
出处
《微生物学报》
CAS
CSCD
北大核心
2013年第7期685-690,共6页
Acta Microbiologica Sinica
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(SKLPBS1112)
江苏大学高级专业人才科研启动基金项目(10JDG044)
江苏省高校自然科学研究项目(12KJD310001)~~
关键词
鼠疫菌
OXYR
DPS
转录调控
Yersinia pestis , OxyR, dps , transcription regulation
