生物技术通报 ›› 2018, Vol. 34 ›› Issue (11): 191-197.doi: 10.13560/j.cnki.biotech.bull.1985.2018-0566

• 研究报告 • 上一篇    下一篇

结核分枝杆菌拟核结合蛋白Lsr2阻遏调控rv1057基因转录的研究

付加芳1, 张佩佩2, 宗工理1, 王新圆2, 刘萌2, 曹广祥1   

  1. 1. 山东省医药生物技术研究中心,济南 250062;
    2. 山东大学 微生物技术国家重点实验室,济南250100
  • 收稿日期:2018-06-20 出版日期:2018-11-26 发布日期:2018-11-28
  • 作者简介:付加芳,女,助理研究员,研究方向:微生物学;E-mail:mefujiafang@163.com
  • 基金资助:
    国家自然科学基金项目(81471921),山东省医学科学院医药卫生科技创新工程项目(201604)

Molecular Mechanism of Nucleoid-Associated Protein Lsr2 Repressing the Expression of Gene rv1057 in Mycobacterium tuberculosis

FU Jia-fang1, ZHANG Pei-pei2, ZONG Gong-li1, WANG Xin-yuan2, LIU Meng2, CAO Guang-xiang1   

  1. 1. Shandong Medicinal Biotechnology Center,Ji’nan 250062;
    2. State Key Laboratory of Microbial Technology,Shandong University,Ji’nan 250100;
  • Received:2018-06-20 Published:2018-11-26 Online:2018-11-28

摘要: 探讨结核分枝杆菌拟核结合蛋白Lsr2是否是rv1057基因转录的阻遏蛋白。利用凝胶阻滞迁移试验分析Lsr2在体外与rv1057启动子片段的特异性结合能力;通过lacZ报告基因和荧光定量PCR检测rv1057启动子区域的不同突变形式对转录的影响;启动转录的能力;通过蛋白质免疫印迹法检测目标基因rv1057的表达。统计学处理采用t检验。Lsr2结合rv1057启动子;Lsr2与已知的阻遏蛋白TrcR都能结合rv1057启动子;缺失Lsr2结合位点的rv1057启动子能够在结核分枝杆菌生长周期中持续启动rv1057基因的转录;野生型菌株H37Rv生长早期(24 h)lsr2基因转录水平较低,而在H37Rv生长中期(48 h)、后期(120 h)lsr2基因的相对表达量变化倍数显著提高,差异具有统计学意义(t值分别为24.44、16.86,P<0.05);H37Rv菌株和lsr2基因缺失菌株中trcR基因在生长早期(24 h)的相对表达量显著高于生长中期(48 h)、生长后期(120 h)的trcR表达量,差异具有统计学意义(t值分别为6.79、10.16,P<0.05)。Lsr2是rv1057基因转录的阻遏蛋白,Lsr2和TrcR均可调控rv1057 基因的表达。Lsr2在结核分枝杆菌生长周期的中后期大量表达并阻遏rv1057的转录,TrcR则在结核分枝杆菌生长周期的早期阻遏rv1057的转录。

关键词: 结核分枝杆菌, rv1057, Lsr2, 阻遏蛋白, 调控

Abstract: The objective of this work is to explore whether or not nucleoid-associated protein Lsr2 of Mycobacterium tuberculosis is a repressor protein in gene rv1057 expression. Electrophoretic mobility shift assay was used to analyze the in vivo specific binding capability of Lsr2 with the target gene promoter region.lacZ report gene and fluorescence quantitative PCR were adapted to detect the effects of different mutations of rv1057 promoter region on the transcription. Western blot was employed to analyze the protein expression level of target gene rv1057. The t test was used for statistical analysis. As results,Lsr2 was able to bind to rv1057 promoter. Both Lsr2 and the known repressor protein TrcR were able to bind to rv1057 promoter. The mutated rv1057 promoter lacking Lsr2 binding site was able to continuously activate the transcription of gene rv1057 during the growth cycle of M. tuberculosis. The relative expression of gene lsr2 was low in the early growth stage(24 h)of wild-type strain H37Rv,while the relative expression of gene lsr2 in H37Rv significantly increased both in the middle growth stage(48 h)and in the later growth stage(120 h),the difference was statistically significant(t=24.44,16.86,respectively;P<0.05). The relative expression of the gene trcR of H37Rv and gene lsr2-deleted strain in the early growth stage(24 h)was significantly higher than in the middle growth period(48 h)and the later growth period(120 h),the difference was statistically significant(t=6.79,10.16,respectively;P<0.05). In conclusion,Lsr2 is the repressor protein of gene rv1057 transcription. Both Lsr2 and TrcR may regulate the expression of rv1057. Lsr2 is mainly expressed in the middle and late growth period of the M. tuberculosis,and then represses the expression of rv1057;while TrcR represses the transcription of rv1057 in the early growth stage of the M. tuberculosis.

Key words: Mycobacterium tuberculosis, rv1057, Lsr2, repressor protein, regulating