PmrA对伤寒沙门菌高渗应激后期基因表达调节浅谈.docVIP

PmrA对伤寒沙门菌高渗应激后期基因表达调节浅谈 　 作者：高宇琳， 张海方， 邹 昕， 杜鸿， 夏秋风， 生秀梅， 徐顺高， 黄新祥 【摘要】 目的： 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： 应用自杀质粒介导的同源重组方法制备伤寒沙门菌pmrA基因缺陷变异株；利用伤寒沙门菌全基因组芯片分析技术，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，并选择部分差异表达基因进行RTRCR验证。结果： 经PCR及序列分析证实，伤寒沙门菌pmrA基因缺陷变异株制备成功；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。结论： PmrA在伤寒沙门菌高渗应激后期的基因表达调节中发挥重要作用。 【关键词】 伤寒沙门菌； PmrA； 高渗应激； 基因芯片 ［Abstract］ Objective: To explore the influence of PmrA on gene expression regulation of S. enterica serover Typhi at later stage of hyperosmotic stress. Methods： The pmrA deleted mutant of S. enterica serovar Typhi was prepared by homologous recombination mediated by suicide plasmid. The hyperosmotic stress environment was simulated by increasing the concentration of NaCl in the LB from 50 mmol/L to 300 mmol/L in vitro, and gene expression profiles of wildtype and pmrA deleted mutant of S. enterica serovar Typhi at later stage of osmotic stress were investigated by S. enterica serovar Typhi geneome microarray analysis. RTPCR was performed to prove the results of microarray in some selected genes. Results： PCR and sequencing analysis demonstrated that the pmrA deleted mutant of S. enterica serovar Typhi had been constructed successfully; gene expression profiles analysis revealed that expression of 81 genes and 22 genes were increased and decreased respectively in the pmrA mutant at later stage of hyperosmotic stress. Conclusion： The regulator, PmrA of S. enterica serover Typhi, was important in genes expression regulation in response to hyperosmotic stress. ［Key words］ S. enterica serover Typhi; PmrA; hyperosmotic stress; microarray 伤寒沙门菌（Salmonella enterica serovar Typhi）是沙门菌属中一种人类严重的肠道致病菌，已成为一种重要的原核生物基因表达与信息调控研究的模式菌。目前在沙门菌和埃希菌（Escherichia coli）中已发现数十种双组分调节系统［1］， PmrAB是其中研究较多的一种，属于经典的二元双组分调节系统［2］。目前研究表明沙门菌PmrAB的活化信号主要有高铁离子（Fe3+）、铝离子（Al3+）、酸性pH（如pH 5.5）、钒酸盐［3-5］和低镁离子（Mg2+）［6］, 低镁离子的活化是通过PhoPQ系统促进pmrD的表达来实现的。 近年来，基因芯片技术的发展为全面、高效观察基因表达提供了一个极佳的策略。我们利用沙门菌基因组寡核苷酸芯片进