H-NS蛋白对副溶血弧菌hcp1的转录调控

Transcriptional regulation of hcp1 by H-NS in Vibrio parahaemolyticus

摘要:

【目的】研究调控子H-NS对副溶血弧菌T6SS1结构蛋白基因hcp1的转录调控机制。【方法】利用Western blot检测Hcp1蛋白在野生株(WT)和hns基因敲除株(Δhns)中表达水平的差异。提取WT和Δhns的总RNA,采用实时定量RT-PCR的方法验证H-NS对hcp1的转录调控关系。进而采用引物延伸实验研究hcp1的转录起始位点,并根据产物的丰度判断H-NS对hcp1的调控关系。PCR扩增hcp1的整个启动子区DNA序列,并纯化His-H-NS蛋白,通过凝胶阻滞实验(EMSA)验证His-H-NS对hcp1启动子区是否具有直接的结合作用。【结果】 Western blot和实时定量RT-PCR结果显示H-NS能抑制hcp1的表达;引物延伸结果显示hcp1只有一个转录起始位点T(-62)(翻译起始位点为+1),且其转录活性是H-NS和σ⁵⁴依赖性的;EMSA实验表明H-NS对hcp1的启动子区具有直接的结合作用。【结论】 H-NS能直接结合到hcp1启动子区而抑制其转录表达。

关键词:副溶血弧菌;H-NS;hcp1;转录调控

Abstract:

[Objective] To study the transcriptional regulation of the structural components Hcp1 by H-NS in Vibrio parahaemolyticus.[Methods] Expression of Hcp1 in the wide-type(WT) strain and hns mutant(Δhns) were detected by Western blot using rabbit anti-Hcp1 polyclonal antibodies. Total RNAs were extracted from WT and Δhns strains. Quantitative RT-PCR was carried out to calculate the transcriptional variation of hcp1 between WT and Δhns strains, and then primer extension assay was used to detect the transcription start site and the promoter activity(the amount of primer extension product) of hcp1 in WT and Δhns. The entire promoter region of hcp1 was amplified by PCR with ExTaq^TM DNA polymerase using WT genomic DNA as the template. The over-expressed His-H-NS was purified under native conditions with nickel loaded HiTrap Chelating Sepharose columns(Amersham). Electrophoretic mobility shift assay(EMSA) was applied to analyze the DNA-binding activity of His-H-NS to hcp1 promoter region in vitro.[Results] Western blot and quantitative RT-PCR results showed that expression of Hcp1 was inhibited by H-NS. The primer extension assay detected only one transcription start site located at 62 bp upstream of hcp1, whose transcription was H-NS and σ⁵⁴-dependent. EMSA result indicated that His-H-NS was able to bind the promoter DNA region of hcp1.[Conclusion] The expression of hcp1 was directly repressed by H-NS in V. parahaemolyticus.

Key words:Vibrio parahaemolyticus;H-NS;hcp1;transcriptional regulation