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

作　　者：张伟[1] 侯书宁 何婷[2] 徐淼[2] 黄新祥[2] 杨瑞馥[3] 周冬生[3] 张义全[2,3] 

机构地区：[1]河北北方学院附属第一医院微生物科,河北张家口075000 [2]江苏大学医学院,江苏镇江212013 [3]北京微生物流行病研究所病原微生物生物安全国家重点实验室,北京100071

出　　处：《微生物学通报》2015年第10期1971-1976,共6页Microbiology China

基　　金：江苏大学高级人才科研启动基金项目(No.14JDG166);国家自然科学基金项目(No.31170127)

摘　　要：【目的】研究副溶血弧菌AphA对vop T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vop T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c DNA,利用实时定量RT-PCR进一步研究AphA对靶基因的调控关系。将vop T的启动子区克隆入p HRP309质粒的β-半乳糖苷酶基因上游,构建Lac Z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vop T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】vop T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vop T转录,且该间接抑制作用与VtrA无关。[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 （△aphA）. Primer extension assay was employed to detect the transcription start site and the promoter activity of vopT in WT, and that in △aphA. Quantitative RT-PCR was also applied to calculate the transcriptional variation of target genes between WT and △aphA. 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 AaphA, respectively, to measure the promoter activity （the β-Galactosidase activity） of vopT in WT and AaphA 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.

关 键 词：副溶血弧菌 转录调控 AphA VOPT 

分 类 号：R378[医药卫生—病原生物学]