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【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 investigate the transcriptional regulation of vop T by AphA of Vibrio parahaemolyticus. 【Method】 The total RNA of wild-type strain (WT) and aphA mutant (ΔaphA) was extracted and the transcriptional start site of vop T was studied by primer extension. The regulation of AphA was determined according to the abundance of the product. The total RNA of WT and ΔaphA were reverse transcribed into c DNA respectively, and the relationship between AphA and target genes was further studied by real-time quantitative RT-PCR. The promoter region of vop T was cloned into the upstream of β-galactosidase gene of p HRP309 plasmid to construct Lac Z recombinant plasmid and the recombinant plasmid was transformed into WT and ΔaphA. Galactosidase activity to determine the difference between AphA regulation of vop T. PCR amplification of target gene DNA sequence of the entire promoter region, and purification of His-AphA protein gel electrophoresis (EMSA) to verify His-AphA target gene promoter region has a direct binding. 【Result】 vop T had only one transcription initiation site A (-86), and its transcriptional activity was indirectly inhibited by AphA. RT-PCR and EMSA results showed that AphA also indirectly inhibited the transcription of vtrA. 【Conclusion】 AphA indirectly inhibits vop T transcription, and this indirect inhibition has nothing to do with VtrA.