摘要
为了探讨副溶血性弧菌拟核相关蛋白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 type Ⅲ secretion 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 in Δhns 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 between Δhns and WT;the promoter DNA regions of target genes were cloned into the upstream of the lacZ reporter gene. The recombinant Lac Z plasmids were then transformed into Δhns and WT, respectively, to measure the β-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 in vitro. 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.
作者
张盈
唐浩
仇越
王丽
张义全
Zhang Ying;Tang Hao;Qiu Yue;Wang Li;Zhang Yiquan(School of Medicine,Jiangsu University,Zhenjiang,212013;The First Hospital Affiliated to Henan University,Kaifeng,475000)
出处
《基因组学与应用生物学》
CAS
CSCD
北大核心
2020年第3期1450-1456,共7页
Genomics and Applied Biology
基金
江苏大学高级专业人才科研启动基金项目(14JDG166,13JDG026)资助。
