鸟分枝杆菌PhoP功能分析及其基因突变株的构建

Characterization of PhoP of Mycobacterium avium and construction of its mutant harboring defective PhoP gene

【目的】对鸟分枝杆菌PhoP的功能进行分析及构建PhoP基因突变株,为深入研究PhoP的调控机制打下基础。【方法】利用PCR扩增出鸟分枝杆菌PhoP DNA结合区(PhoPC)编码序列,与表达载体p GEX-4T-3连接后,转化入大肠杆菌BL21(DE3)中表达GST-PhoPC融合蛋白。用凝血酶去除GST标签,制备PhoPC蛋白;利用PCR扩增出鸟分枝杆菌PhoP基因及其下游基因MAV0127、PhoU和Amt的启动子片段,采用凝胶迁移率移动试验(EMSA)分别检测PhoPC与PhoP、MAV0127、PhoU和Amt的启动子结合的情况。通过PCR扩增PhoP基因上、下游片段,构建PhoP基因缺失性同源核苷酸片段,与自杀质粒p GMB151连接后,通过电转化导入鸟分枝杆菌进行同源交换,利用PCR筛选出PhoP基因缺失突变株。【结果】EMSA结果显示,鸟分枝杆菌PhoP能与PhoP、MAV0127及Amt基因启动子结合,不能与PhoU结合。通过PCR和序列分析证实基因突变株的PhoP基因缺失了309个碱基。【结论】PhoP不仅可调控其下游基因MAV0127和Amt的转录水平,还可调控其自身基因的转录,但不参与调节PhoU二元调控系统。构建了PhoP基因缺失突变株,为进一步研究其在鸟分枝杆菌的调控功能奠定了基础。

[Objective] To understand the regulatory mechanism of PhoP of Mycobacterium avium, the function of PhoP was analyzed and a Mycobacterium avium strain with defective PhoP gene was constructed. [Methods] DNA fragment of PhoP DNA-binding domain（PhoPC） amplified by PCR was cloned into the p GEX-4T-3 vector, and then the recombinant plasmid was transformed into Escherichia coli BL21（DE3） for expression of GST-PhoPC protein. PhoPC protein was prepared through removal of GST-tag from GST-PhoPC protein by thrombin cleaving. The promotor regions of PhoP, MAV0127, PhoU and Amt were amplified by PCR, respectively. Electrophoretic mobility shift assay（EMSA） was carried out to analyze the binding activity of PhoPC to these promotor fragments. For construction of a Mycobacterium avium strain with defective PhoP gene, PhoP homologous recombinant DNA fragment with defective mutation was obtained by ligasing the upper-stream and down-stream regions of PhoP gene, which were amplified by PCR. The PhoP recombinant DNA fragment was cloned into the suicide plasmid p GMB151. The recombinant plasmid was then transferred into cells of Mycobacterium avium by using electroporation for homologous recombination. The strains harboring defective PhoP gene were selected and identified by PCR. [Results] EMSA results show that PhoPC protein could bind to the promotors of PhoP, MAV0127 and Amt genes, but not bind to the promotor of PhoU. A deletion of 309 bp of the defective PhoP gene was confirmed by PCR and DNA sequencing. [Conclusion] PhoP can regulate the transcription of its downstream genes MAV0127 and Amt, and also regulate the transcription of PhoP gene itself. However, PhoP does not participate in regulating PhoU two-component system. A Mycobacterium avium strain with defective PhoP gene was successfully constructed, which contributes to further study on the role of PhoP in transcription regulation in Mycobacterium avium.