副溶血性弧菌QS系统核心调控子OpaR和AphA对mfp和cpsQ的调控研究

发布时间：2018-05-14 05:21

  本文选题：副溶血性弧菌 + 转录调控　； 参考：《重庆医科大学》2013年硕士论文

【摘要】：背景副溶血性弧菌是一种食源性致病菌，具有较强的生物膜形成能力，其在形成生物膜过程中，主要受QS系统调节。OpaR和AphA是副溶血性弧菌QS系统的两个核心调控子，在不同的菌密度条件下调控生物膜的形成。cpsQ和mfp基因座位紧密连锁，两者均与细胞表面特征有关，其转录受多个调控子的调控，如cpsQ的转录表达受CpsR的调控和CpsQ自调控。那么，OpaR和AphA对cpsQ和mfp是否存在直接调控关系呢？还需进一步验证。 目的深入研究副溶血性弧菌QS系统核心调控子OpaR和AphA对生物膜相关基因cpsQ和mfpA的调控机制。 方法利用自杀载体的方法构建副溶血性弧菌opaR和aphA基因的无痕非极性突变株；提取野生株与调控子基因突变株在特定培养条件下的总RNA，利用引物延伸实验研究mfpA与cpsQ的转录起始位点，并根据引物延伸条带的相对丰度，判定调控子蛋白对靶基因的调控关系；PCR扩增mfpA与cpsQ的整个启动子区DNA序列，纯化回收后，将其直接克隆入pHRB309质粒无启动子区β-半乳糖苷酶基因的上游，将重组质粒分别转入副溶血性弧菌野生株和调控子基因突变株中，通过测定二者中β-半乳糖苷酶活性差异，进一步验证调控子对靶基因的调控关系；分别扩增mfpA与cpsQ的整个启动子区DNA序列，并表达纯化副溶血弧菌His-OpaR与His-AphA重组蛋白，通过凝胶阻滞实验（EMSA）验证调控子蛋白对靶基因启动子区是否具有直接的相互作用；最后通过DNaseⅠ足迹实验，验证调控子蛋白对靶基因启动子区具体相互作用的位点。 结果AphA低密度表达，只有一个转录起始位点C（-200），其转录起始受密度抑制。OpaR高密度表达，也只有一个转录起始位点G（-74），其转录具有密度依赖性。低密度条件下： mfpA只有一个转录起始位点A（-70）（翻译起始位点为+1），其转录起始受AphA的抑制；体外实验表明，AphA能直接结合到mfpA启动子区-84和-129之间的碱基序列上。cpsQ也只有一个转录起始位点C（-70），且其转录起始也同样受AphA的抑制，，但这种抑制是间接的。高密度条件下：mfpA和cpsQ的转录起始受OpaR的激活，其转录起始位点均同低密度条件。体外实验表明，OpaR能直接结合到mfpA启动子区-133和-177之间的碱基序列上，却不能直接与cpsQ结合，因此其对mfpA的激活是直接的，而对cpsQ的激活是间接的。mfpA和cpsQ基因的转录也是密度依赖性的，均只有一个转录起始位点，分别为A（-70）和C（-70）。 结论在低菌密度条件下，AphA大量表达，进而直接抑制mfpA基因的转录，但间接抑制cpsQ的转录；在高菌密度条件下，OpaR大量表达，进而直接激活mfpA基因的转录，但间接激活cpsQ基因的转录；mfpA和cpsQ基因的转录与opaR和aphA基因一样，也受菌密度的调节，二者均在高密度下高表达。
[Abstract]:Background Vibrio parahaemolyticus is a foodborne pathogenic bacterium with strong biofilm forming ability. During the biofilm formation, it is mainly regulated by QS system. OpaR and AphA are two core regulators of QS system of Vibrio parahaemolyticus. The biofilm formation. CPQ and mfp gene loci are closely linked to cell surface characteristics under different bacterial densities, and their transcription is regulated by multiple regulators, such as CpsR and CpsQ self-regulation. So is there a direct regulatory relationship between OpaR and AphA on cpsQ and mfp? Further verification is required. Objective to investigate the regulatory mechanisms of OpaR and AphA on biofilm-associated genes cpsQ and mfpA in Vibrio parahaemolyticus QS system. Methods Non-polarity mutant strains of vibrio parahaemolyticus opaR and aphA genes were constructed by suicide vector. The total RNAs of wild and regulatory gene mutants were extracted under specific culture conditions. The transcriptional initiation sites of mfpA and cpsQ were studied by primer extension experiment, and the relative abundance of the bands was determined according to the primer extension. The whole promoter region DNA sequence of mfpA and cpsQ was amplified by PCR and cloned into the upstream of 尾 -galactosidase gene of pHRB309 plasmid. The recombinant plasmids were transferred into wild vibrio parahaemolyticus strain and mutant of regulator gene respectively. The difference of 尾 -galactosidase activity between them was determined to further verify the regulatory relationship between the regulator and the target gene. The whole promoter DNA sequence of mfpA and cpsQ was amplified, and the recombinant protein of His-OpaR and His-AphA was expressed and purified from Vibrio parahaemolyticus. Finally, the specific interaction sites of regulatory proteins on target gene promoter region were verified by DNase 鈪

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