双组份信号转导系统MprAB和TrcRS协同调控结核分枝杆菌rv1057基因表达的分子机制

The molecular mechanism of two-component system of MprAB and TrcRS in synergistically regulating gene rv1057 expression of Mycobacterium tuberculosis

目的探讨结核分枝杆菌rv1057基因受到双组份信号转导系统MprAB和TrcRS协同调控的作用机制。方法采用凝胶阻滞迁移试验分析MprA和TrcR在体外与目标基因启动子片段的特异性结合能力；荧光定量PCR检测目标基因的转录水平，以H37RvSDS处理组的表达值为1个单位进行倍数变化分析；蛋白质免疫印迹法检测目标基因的表达；通过构建lacZ报告基因检测rv1057启动子不同区域启动转录的能力。统计学处理采用t检验。结果MprA结合trcR启动子；无SDS条件下D981、H37Rv菌株的trcR相对表达量分别为H37RvSDS处理组的1．7和2．5倍，差异有统计学意义（t=18．54，P〈0．05）；有SDS条件下，D981、H37Rv菌株的trcR相对表达量分别为H37RvSDS处理组的1．0和2．1倍，D981菌株的trcR转录水平显著高于H37Rv菌株，差异有统计学意义（t=15．86，P〈0．05）。H37Rv菌株培养过程中加入SDS后trcR的相对表达量变化倍数显著下降，差异有统计学意义（￡一16．99，P〈0．05）；MprA和TrcR都可以结合rv1057启动子并调控其表达；MprA激活rv1057的转录，而TrcR阻遏rv1057的转录。结论MprAB和TrcRS协同调控rv1057基因的表达，环境存在SDS时MprA被激活，阻碍trcR表达并激活r口1057的转录，而无sDs时TrcR阻遏rv1057的转录。

Objective To study the mechanism of two-component system of MprAB and TrcRS in synergistically regulating gene rv1057 expression of Mycobacterium tuberculosis. Methods The in vivo specific binding capability of MprA and TrcR with the target gene promoter region was analyzed using electrophoretic mobility shift assay. The transcription level of target gene was analyzed by using fluorescence quantitative polymerase chain reaction, and all results were compared with the fold changes in H37Rv strain plus SDS group, which was set as one unit. The expression level of target gene was analyzed by using western blot; the transcription ability of different promoter region of rv1057 was detected through lacZ report gene. The t test was used for statistical analysis. Results MprA was able to bind to trcR promoter. The expressions of trcR in D981 and H37Rv strains without SDS were 1.7 and 2.5 folds of the expression of H37Rv strains with SDS groups, respectively. The difference between these two groups was statistically significant （t= 18.54, P^0.05）. With SDS, the expressions of trcR in D981 and H37Rv strains were 1.0 and 2.1 folds of the expression of H37Rv strains plus SDS group, respectively. The expressions of trcR in D981 and H37Rv strains were significantly different （t= 15.86, P^0.05）.After adding SDS during the culture of H37Rv strains, the expression of trcR in H37Rv decreased. The difference between these two groups was statistically significant （t= 16.99, P=0.05）. Both MprA and TrcR were able to bind to rv1057 promoter and regulate its expression. MprA activated the expression of rv1057, while TrcR repressed the expression of rv1057. Conclusions MprAB and TrcRS synergistically regulate the expression of rv1057. MprA is activated in the presence of SDS, which represses the transcription of trcR and activates the transcription of rv1057. However, TrcR represses the transcription of rv1057 in the absence of SDS.