PmrA对伤寒沙门菌高渗应激后期基因表达调节作用

发布时间：2018-01-07 07:24

  本文关键词：PmrA对伤寒沙门菌高渗应激后期基因表达调节作用　出处：《江苏大学》2009年硕士论文　论文类型：学位论文

  更多相关文章： 伤寒沙门菌 PmrA 基因芯片 高渗应激 侵袭

【摘要】： 目的 PmrA为伤寒沙门菌双组份调节系统的效应调节蛋白,在高渗应激的后期其基因表达明显下调。本研究的目的是进一步探讨伤寒沙门菌调节因子PmrA在高渗应激后期对基因表达调节的影响。 方法 (1)伤寒沙门菌pmrA基因缺陷变异株制备:采用自杀质粒pGMB151介导的同源重组方法制备伤寒沙门菌调节因子PmrA的基因缺陷变异株,根据伤寒沙门菌pmrA基因序列设计引物,扩增pmrA基因上、下游同源性片段,定向连接成pmrA基因缺损型同源核苷酸片段,并与自杀质粒pGMB151相连后经电击法导入伤寒沙门菌野生株,在5%蔗糖LB平板上进行同源重组,通过筛选获得pmrA基因缺陷变异株; (2)伤寒沙门菌基因转录表达谱分析:利用伤寒沙门菌全基因组芯片分析技术,体外模拟高渗环境应激,在低渗(50 mmol/L NaCl)LB培养液中分别培养伤寒沙门菌野生株和pmrA基因缺陷变异株4h(37℃,250r/min)至对数生长期,后转入高渗(300 mmol/L NaCl)LB培养液中在同样条件下继续培养,在高渗应激后期(120min),分别提取伤寒沙门菌野生株和pmrA基因缺陷变异株的总RNA,反转录成cDNA并标记荧光(cy3或cy5),与基因组芯片杂交,扫描后据荧光信号分析各基因转录表达水平,比较伤寒沙门菌野生株和pmrA基因缺陷变异株在高渗应激后期的基因表达谱差异; (3)实时荧光定量PCR(qRT-PCR)分析:选择部分表达差异显著的基因,设计并合成特异性引物,进行实时荧光定量PCR,验证DNA芯片分析结果。, 结果 (1)PCR及序列分析证实,pmrA基因缺陷变异株中pmrA基因351bp被6bp取代,表明成功构建pmrA基因缺陷变异株; (2)基因表达谱比较分析结果表明伤寒沙门菌pmrA基因缺陷变异株在高渗应激后期有81个基因表达上调,有22个基因表达下调。其主要涉及ABC转运系统相关蛋白、鞭毛蛋白、侵袭蛋白、外膜蛋白及一些酶类等; (3)对pmrA基因缺陷变异株中表达差异基因(yliC,putA),利用实时荧光定量PCR进行分析其mRNA水平,结果显示其在pmrA基因缺陷变异株和野生株中差异与基因芯片分析结果基本一致。 结论 成功构建伤寒沙门菌pmrA基因缺陷变异株;PmrA在伤寒沙门菌高渗应激后期,对基因表达调节与物质转运及侵袭力的提高等发挥重要作用。
[Abstract]:objective
PmrA is the regulation protein of two components of Salmonella typhi, and its gene expression is down regulated at the late stage of hyperosmotic stress. The purpose of this study is to further explore the effect of Salmonella typhimurium regulatory factor PmrA on gene expression regulation in the late stage of hyperosmotic stress.
Method
(1) pmrA gene deleted mutant of Salmonella typhi was prepared by Dutch act plasmid mediated pGMB151 of Salmonella typhi regulator PmrA homologous recombination method for the mutant, the primers were designed according to the Salmonella pmrA gene sequence, pmrA gene amplification, downstream homologous fragment, connection oriented pmrA gene defect type homologous nucleotide fragment and plasmid pGMB151, and Dutch act after electroporation is connected into the wild strains of Salmonella typhi, homologous recombination in 5% sucrose LB tablet, obtained by pmrA mutant screening;
(2) the expression of Salmonella typhi gene transcription analysis: spectrum analysis technique using Salmonella typhi genome gene chip. The in vitro hypertonic environment stress in low permeability (50 mmol/L NaCl) were cultured in LB culture of Salmonella typhimurium wild strain and the pmrA mutant 4H solution (at 37 250r/min) to the logarithmic growth phase, then transferred to hypertonic (300 mmol/L NaCl) in LB medium under the same conditions continue to develop, in the later stage of hyperosmotic stress (120min), total RNA was extracted from Salmonella typhimurium wild-type and pmrA mutant, reverse transcription cDNA and labeled (Cy3 or Cy5), and hybridized with the gene chip, according to fluorescence scanning signal analysis of each gene expression, comparison of Salmonella typhimurium wild strain and the pmrA mutant at later stage of hyperosmotic stress the difference of gene expression profile;
(3) real-time fluorescence quantitative PCR (qRT-PCR) analysis: select partial genes that express significant differences, design and synthesize specific primers, real-time fluorescence quantitative PCR, verify the results of DNA chip analysis.
Result
(1) PCR and sequence analysis confirmed that the pmrA gene 351bp in the pmrA gene defective mutant was replaced by 6BP, indicating that the pmrA gene defective mutant was successfully constructed.
(2) expression profiles analysis revealed that pmrA gene deleted mutant of Salmonella typhi at later stage of hyperosmotic stress expression of 81 genes, 22 genes were down regulated. It mainly involves the related protein, ABC transporter flagellin, invasion protein, membrane protein and enzymes;
(3) differentially expressed genes (yliC, putA) in pmrA gene variant mutant were analyzed by real-time fluorescence quantitative PCR. The results showed that the difference between pmrA gene deficient mutant and wild strain was basically the same with that of gene chip analysis.
conclusion
We successfully constructed pmrA gene deficient mutant of Salmonella typhi. PmrA played an important role in the regulation of gene expression, transport and invasion of Salmonella typhi in the late stage of hyperosmotic stress.

【学位授予单位】：江苏大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R378.23

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