结核分枝杆菌Mtub-39位点多态性与下游基因表达关系的研究

Research on relationship between polymorphisms of Mtub-39 and expression of downstream genes of Mycobacterium tuberculosis

目的分析结核分枝杆菌(MTB)北京型菌株及非北京型菌株5个有明显差异的MTB散在分布重复单位(MIRU)位点,研究其多态性与下游基因表达的关系。方法利用生物信息学方法预测5个MIRU位点(ETR-C、Mtub-30、Mtub-39、MIRU-27、MIRU-40)下游相邻基因启动子区域。通过PCR方法扩增该区域并与分枝杆菌启动子探针载体pMC210连接,重组质粒测序正确后,用电穿孔方法转化耻垢分枝杆菌mc2155。采用Real-Time PCR技术检测报告基因lacZ的转录水平,验证MIRU位点多态性对其下游基因表达的影响。结果 Mtub-39位点核心区域内含有其下游基因的启动子。成功构建了分别带有1、3、5个拷贝重复序列的Mtub-39位点的重组质粒。Real-Time PCR结果显示,有多态性的Mtub-39位点(pMC210-Mtub-39-N158、pMC210-Mtub-39-N139及pMC210-Mtub-39-N146)对报告基因lacZ的转录调控差异有统计学意义(P=0.006 5)。结论 Mtub-39位点的多态性能明显影响下游基因启动子的活性进而调节其表达。

Objective To analyse the 5 MIRU loci of Mycobacterium tuberculosis, and investigate the relationship between polymorphisms of MIRU loci and expression of downstream genes. Methods Bioinformatics method was used to predict the downstream genes promoter regions of 5 MIRU loci （ETR-C, Mtub-30, Mtub-39, MIRU-27 and MIRU-40）. Promoter sequence was amplified by PCR, and was cloned into mycobacterial promoterless probe vector pMC210 to generate the recombinants. After confirmation by restriction endonuclease digestion and sequence analysis, the recombinant plasmids were transformed into mycobacterium smegmatis mc~ 155 by electroporation. The transcriptional level of reporter gene lacZ was evaluated by Real-Time PCR, and the influence of polymorphisms of MIRU loci on the expression of downstream genes was examined. Results Mtub-39 locus core region contained the promoter of the downstream gene. The recombinant plasmids harboring Mtub-39 locus with 1, 3 or 5 copy numbers were constructed. Real-Time PCR revealed that Mtub-39 locus with polymorphisms （pMC210-Mtub-39-N158, pMC210-Mtub-39-N139 and pMC210-Mtub-39-N146） had significant differences in the transcriptions of reporter gene lacZ （P = 0. 006 5）. Conclusion The polymorphisms of Mtub-39 can significantly affect the promotor activity of the downstream genes, and sequentially regulate the expression of the gene.