副溶血弧菌拟核相关蛋白H-NS间接抑制VP1687-1686的启动子区转录

The Nucleoid-Associated Protein H-NS in Vibrio parahaemolyticus Indirectly Inhibits the Transcription of VP1687-1686 Operon Promoter Region

为了探讨副溶血弧菌拟核相关蛋白H-NS对Ⅲ型分泌系统(T3SS)VP1687-1686基因位点的转录调控,本研究提取副溶血弧菌hns突变株(Δhns)和野生株(WT)的总RNA,采用引物延伸实验研究靶基因的转录起始位点,并根据产物的丰度判断H-NS对靶基因的调控关系;采用实时定量RT-PCR研究靶基因mRNA在WT和Δhns中转录丰度,以判定H-NS对靶基因的转录调控关系;将靶基因启动子区域DNA序列克隆至lacZ基因上游,将重组质粒转入WT和Δhns中,得到相应的LacZ菌株,通过LacZ报告基因融合实验研究H-NS对靶基因的调控关系;用PCR扩增靶基因的启动子区DNA序列,并纯化His-H-NS蛋白,通过凝胶阻滞实验(EMSA)研究His-H-NS是否对靶基因启动子区具有直接的结合作用。研究结果显示,T3SS的VP1687-1686只含有一个转录起始位点,位于翻译起始位点上游82 bp处,且H-NS能够抑制其转录活性,但不能直接结合到VP1687-1686区的启动子区。另外,H-NS对calR的转录无调控作用,His-H-NS也不能结合到其启动子区。本研究的结果初步说明,H-NS能够间接抑制VP1687-1686的转录,该抑制机制与CalR无关联。

In order to investigate the transcriptional regulation of the typeⅢsecretion system(T3SS)VP1687-1686 operon by nucleoid-associated protein H-NS in Vibrio parahaemolyticus,total RNAs were extracted from the hns null mutant(Δhns)and wild-type(WT)strains.The primer extension assay was employed to detect the transcription start sites of target genes.The regulation of H-NS on target genes was determined according to the abundance of primer extension products of target genes inΔhns and WT;To further determine the transcriptional regulation of H-NS on target genes,quantitative RT-PCR was carried out to calculate the transcriptional variation of target genes betweenΔhns and WT;The promoter DNA regions of target genes were cloned into the upstream of the lacZ reporter gene.The recombinant LacZ plasmids were then transformed intoΔhns and WT,respectively,to measure theβ-galactosidase activities;The DNA sequences in the promoter region of the target genes were amplified by PCR and the over-expressed His-H-NS was purified under native conditions.The electrophoretic mobility shift assay(EMSA)was applied to analyze the DNA-binding activity of His-H-NS to target promoters invitro.The results showed that a single transcription start site for T3SS VP1687-1686,which was located 82 bp upstream of the translation start site,and its transcribed activity was under the negative control of the H-NS.However,H-NS was unable to bind to the promoter region of VP1687-1686.Moreover,H-NS has no binding activity and regulatory effect on calR transcription.The results of this study preliminarily indicated that H-NS in Vibrio parahaemolyticus repressed the transcription of VP1687-1686 in an indirect manner,which was not associated with CalR.