本文選題：副溶血性弧菌 + 轉(zhuǎn)錄調(diào)控�。� 參考：《重慶醫(yī)科大學(xué)》2013年碩士論文

【摘要】：背景副溶血性弧菌是一種食源性致病菌，具有較強(qiáng)的生物膜形成能力，其在形成生物膜過程中，主要受QS系統(tǒng)調(diào)節(jié)。OpaR和AphA是副溶血性弧菌QS系統(tǒng)的兩個(gè)核心調(diào)控子，在不同的菌密度條件下調(diào)控生物膜的形成。cpsQ和mfp基因座位緊密連鎖，兩者均與細(xì)胞表面特征有關(guān)，其轉(zhuǎn)錄受多個(gè)調(diào)控子的調(diào)控，如cpsQ的轉(zhuǎn)錄表達(dá)受CpsR的調(diào)控和CpsQ自調(diào)控。那么，OpaR和AphA對cpsQ和mfp是否存在直接調(diào)控關(guān)系呢？還需進(jìn)一步驗(yàn)證。 目的深入研究副溶血性弧菌QS系統(tǒng)核心調(diào)控子OpaR和AphA對生物膜相關(guān)基因cpsQ和mfpA的調(diào)控機(jī)制。 方法利用自殺載體的方法構(gòu)建副溶血性弧菌opaR和aphA基因的無痕非極性突變株；提取野生株與調(diào)控子基因突變株在特定培養(yǎng)條件下的總RNA，利用引物延伸實(shí)驗(yàn)研究mfpA與cpsQ的轉(zhuǎn)錄起始位點(diǎn)，并根據(jù)引物延伸條帶的相對豐度，判定調(diào)控子蛋白對靶基因的調(diào)控關(guān)系；PCR擴(kuò)增mfpA與cpsQ的整個(gè)啟動(dòng)子區(qū)DNA序列，純化回收后，將其直接克隆入pHRB309質(zhì)粒無啟動(dòng)子區(qū)β-半乳糖苷酶基因的上游，將重組質(zhì)粒分別轉(zhuǎn)入副溶血性弧菌野生株和調(diào)控子基因突變株中，通過測定二者中β-半乳糖苷酶活性差異，進(jìn)一步驗(yàn)證調(diào)控子對靶基因的調(diào)控關(guān)系；分別擴(kuò)增mfpA與cpsQ的整個(gè)啟動(dòng)子區(qū)DNA序列，并表達(dá)純化副溶血弧菌His-OpaR與His-AphA重組蛋白，通過凝膠阻滯實(shí)驗(yàn)（EMSA）驗(yàn)證調(diào)控子蛋白對靶基因啟動(dòng)子區(qū)是否具有直接的相互作用；最后通過DNaseⅠ足跡實(shí)驗(yàn)，驗(yàn)證調(diào)控子蛋白對靶基因啟動(dòng)子區(qū)具體相互作用的位點(diǎn)。 結(jié)果AphA低密度表達(dá)，只有一個(gè)轉(zhuǎn)錄起始位點(diǎn)C（-200），其轉(zhuǎn)錄起始受密度抑制。OpaR高密度表達(dá)，也只有一個(gè)轉(zhuǎn)錄起始位點(diǎn)G（-74），其轉(zhuǎn)錄具有密度依賴性。低密度條件下： mfpA只有一個(gè)轉(zhuǎn)錄起始位點(diǎn)A（-70）（翻譯起始位點(diǎn)為+1），其轉(zhuǎn)錄起始受AphA的抑制；體外實(shí)驗(yàn)表明，AphA能直接結(jié)合到mfpA啟動(dòng)子區(qū)-84和-129之間的堿基序列上。cpsQ也只有一個(gè)轉(zhuǎn)錄起始位點(diǎn)C（-70），且其轉(zhuǎn)錄起始也同樣受AphA的抑制，，但這種抑制是間接的。高密度條件下：mfpA和cpsQ的轉(zhuǎn)錄起始受OpaR的激活，其轉(zhuǎn)錄起始位點(diǎn)均同低密度條件。體外實(shí)驗(yàn)表明，OpaR能直接結(jié)合到mfpA啟動(dòng)子區(qū)-133和-177之間的堿基序列上，卻不能直接與cpsQ結(jié)合，因此其對mfpA的激活是直接的，而對cpsQ的激活是間接的。mfpA和cpsQ基因的轉(zhuǎn)錄也是密度依賴性的，均只有一個(gè)轉(zhuǎn)錄起始位點(diǎn)，分別為A（-70）和C（-70）。 結(jié)論在低菌密度條件下，AphA大量表達(dá)，進(jìn)而直接抑制mfpA基因的轉(zhuǎn)錄，但間接抑制cpsQ的轉(zhuǎn)錄；在高菌密度條件下，OpaR大量表達(dá)，進(jìn)而直接激活mfpA基因的轉(zhuǎn)錄，但間接激活cpsQ基因的轉(zhuǎn)錄；mfpA和cpsQ基因的轉(zhuǎn)錄與opaR和aphA基因一樣，也受菌密度的調(diào)節(jié)，二者均在高密度下高表達(dá)。
[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

本文編號(hào)：1886528