AphA蛋白对副溶血弧菌vopT的转录调控研究

Transcriptional regulation of vopT by AphA in Vibrio parahemolyticus

摘要:

【目的】研究副溶血弧菌AphA对vopT的转录调控机制。【方法】提取野生株(WT)和aphA突变株(ΔaphA)的总RNA，采用引物延伸实验研究vopT的转录起始位点，并根据产物的丰度差异判断AphA对其调控关系。分别将WT和ΔaphA的总RNA逆转录成cDNA，利用实时定量RT-PCR进一步研究AphA对靶基因的调控关系。将vopT的启动子区克隆入pHRP309质粒的β-半乳糖苷酶基因上游，构建LacZ重组质粒，并将该重组质粒转入WT和ΔaphA中，通过测定并比较两株菌中β-半乳糖苷酶活性的差异来判定AphA对vopT的调控关系。PCR扩增靶基因整个启动子区DNA序列，并纯化His-AphA蛋白，利用凝胶阻滞实验(EMSA)验证His-AphA对靶基因启动子区是否具有直接的结合作用。【结果】vopT只有一个转录起始位点A (?86)，且其转录活性受AphA的间接抑制。RT-PCR和EMSA结果显示AphA对vtrA的转录也具有间接的抑制作用。【结论】AphA间接抑制vopT转录，且该间接抑制作用与VtrA无关。

Abstract:

[Objective] To study the transcriptional regulation of vopT by AphA in Vibrio parahemolyticus. [Methods] Total RNAs were extracted from the wide-type (WT) strain and the aphA mutant (ΔaphA). Primer extension assay was employed to detect the transcription start site and the promoter activity of vopT in WT, and that in ΔaphA. Quantitative RT-PCR was also applied to calculate the transcriptional variation of target genes between WT and ΔaphA. The entire promoter region of vopT was cloned into the pHRP309 containing a promoterless lacZ gene. The recombinant lacZ reporter plasmid was transformed into WT and ΔaphA, respectively, to measure the promoter activity (the β-Galactosidase activity) of vopT in WT and ΔaphA by using the β-Galactosidase Enzyme Assay System. The over-expressed His-AphA was purified under native conditions with nickel loaded HiTrap Chelating Sepharose columns (Amersham), and the entire promoter region of the target genes was amplified by PCR. Then, the electrophoretic mobility shift assay (EMSA) was applied to analyze the DNA-binding activity of His-AphA to target promoter regions in vitro. [Results] Primer extension assay detected only one transcriptional start site located at 86 bp upstream of vopT, whose transcript was negative regulated by AphA in an indirectly manner. Moreover, RT-PCR and EMSA results showed that the transcription of vtrA was also indirectly controlled by AphA. [Conclusion] AphA repressed the transcription of vopT indirectly, and this indirect inhibition was not dependent on VtrA.