【作者】 夏秋风；

【导师】 黄新祥；

【作者基本信息】 江苏大学 ， 临床检验诊断学， 2009， 硕士

【摘要】 目的Mig-14是伤寒沙门菌的一种内膜结合蛋白,在高渗应激时期表达显著增高,蛋白结构分析提示它可能为一种调节蛋白。本研究目的是揭示伤寒沙门菌Mig-14在高渗应激下对基因表达的调节作用。方法1.用自杀质粒介导的同源重组方法,制备伤寒沙门菌mig-14基因缺陷变异株。根据伤寒沙门菌mig-14基因序列,设计PCR特异性引物,并在引物5’末端加上需要的酶切位点。扩增mig-14基因上、下游DNA片段,用BglⅡ消化后,定向连接成mlg-14基因的缺损性同源性核苷酸片段。将此片段胶回收后克隆至自杀质粒pGMB151的BamH I位点,再将经酶切和特异性PCR鉴定的阳性重组自杀质粒用电击法导入伤寒沙门菌野生株,用蔗糖诱导同源重组。用PCR观察重组现象,将完全重组的菌株作为mig-14基因缺陷变异株,并通过相应的核苷酸序列分析加以确证。2.利用伤寒沙门菌全基因组芯片分析技术,在体外模拟高渗应激环境,向在低渗培养基中培养至对数生长期的菌液中加入NaCl,使其浓度从50 mM增加到300 mM。在高渗应激早期(30min)分别提取伤寒沙门菌野生株和mig-14基因缺陷变异株总RNA,反转录成cDNA并加标荧光(cy3或cy5),与基因组芯片进行杂交,扫描分析,比较伤寒沙门菌野生株和mig-14基因缺陷变异株在高渗应激早期的基因表达谱差异,并选择部分差异表达基因利用实时荧光定量RT-PCR验证,以分析Mig-14在高渗应激下对基因表达的调节作用。3.在中性和酸性条件下对mig-14缺陷株、phoP缺陷株和野生株做多粘菌素B杀伤实验,以确证在S.Typhi GIFU10007野生株中mig-14是否能拮抗多粘菌素B的杀伤作用。结果1.成功制备伤寒沙门菌mig-14缺陷株,经PCR及序列分析证实,mig-14变异株中mig-14基因的编码区有390个碱基缺失,且没有发生极性突变。2.基因表达谱比较分析结果表明,在高渗应激早期mig-14变异株与野生株相比有77个基因表达下调和72个基因表达上调,其中包括鞭毛蛋白、侵袭蛋白、外膜蛋白、代谢酶类以及一些未知功能的蛋白。选取其中部份差异表达基因cydA、invF、pagP和treC,利用实时荧光定量RT-PCR进行验证,结果显示所选基因的mRNA水平与基因芯片分析的结果情况一致。3.在中性培养基条件下,多粘菌素B对三种菌株的杀伤效果基本相同。在酸性条件下mig-14缺陷株确实对多粘菌素B的敏感性比野生株稍高,但敏感程度比phoP缺陷株低很多。结论伤寒沙门菌Mig-14为一种内膜结合的调节蛋白,在高渗应激早期主要参与调节鞭毛装配过程以及细菌的物质和能量代谢。更多还原

【Abstract】 ObjectivePrevious researches revealed that Mig-14 is an inner membrane binding protein and a putative regulator protein.Microarray analysis of global gene expression profiles during hyperosmotic stress in Salmonella enterica serovar Typhi(S.Typhi) showed that the expression of mig-14 is increased at early stage of the hyperosmotic stress.The goal of the study is to clarify the function of Mig-14 on gene expression regulation at early stage of the hyperosmotic stress in S.Typhi.MethodsThe mig-14 deleted mutant ofS.Typhi was prepared by homologous recombination mediated by suicide plasmid pGMB 151.As the genomic information,two pairs of primers were designed to amplify two homologous DNA fragments(upper-and down-stream fragments of the mig-14 gene of S.Typhi) that were ligated by orientational connection as the homologous recombinant DNA fragment,which was then inserted into the suicide plasmid pGMB151 BamH I site.The positive recombinant plasmid was selected by specific PCR and digested with BamH I.The wild-type strain of S.Typhi was then transformed with the recombinant plasmid by electroporation.The mutant mig-14~- was selected by screening on the LB sucrose plate and selected by specific PCR and finally verified by the relative DNA sequencing.The hyperosmotic stress environment was simulated by an osmotic up-shift,which increased the concentration of NaCl in the LB from 50 mM to 300 mM in vitro,the total RNA was extracted from wild-type strain and mig-14 deleted mutant of S.Typhi,respectively,at early stage of the hyperosmotic stress (30min).The gene expression profiles of wild-type and mig-14 deleted mutant of S.Typhi at early stage of the hyperosmotic stress were investigated with a genomic DNA microarray,qRT-PCR was performed for some selected genes which had significantly different expression in the mig-14 deleted mutant to prove the results of microarray.The polymyxin B sensitivity assay was performed for wild-type strain,mig-14 mutant and phoP mutant at neutral and poor acid environment respectively in order to compare their resistance to polymyxin B.ResultsPCR and sequencing analysis showed that the 390 bp in the encoding region of the mig-14 gene was deleted,and demonstrated that the mig-14 gene deleted mutant or S.enterica serovar Typhi without polar mutation was constructed successfully.Gene expression profiles analysis revealed that expression of 72 genes and 77 genes were induced and decreased,respectively,in the mig-14 mutant at early stage of hyperosmotic stress.The regulated genes were involved in flagellar proteins,invasion proteins,outer membrane proteins,metabolic enzymes and some other unkown function proteins.Expressions of genes(cydA, invF,pagP and treC) which have significantly different expression in the mig-14 deleted mutant were investigated by qRT-PCR,and the results showed that the expressions of them are consistent with the results from the microarray assay.ConclusionsThe mig-14 deleted nonpolar mutant of S.Typhi was constructed successfully.The inner membrane regulator Mig-14 of S.Typhi is important to genes expression regulation in response to the hyperosmotic stress,which involves in the assembly of flagella and the substance metabolism and the energy metabolism of S.enterica.There was nearly no difference among the sensitiveness of the three kinds of S.Typhi strain at neutral environment.However at poor acid environment,mig-14 mutant was more and less sensitive to polymyxin B than wild-type strain and phoP mutant respectively from the polymyxin B sensitivity assay.更多还原