UhpA对伤寒沙门菌高渗应激的基因表达调节作用
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摘要
目的
UhpA为伤寒沙门菌双组份调节系统的效应调节蛋白,在高渗应激下其基因表达明显上调。本研究的目的是进一步探讨伤寒沙门菌调节因子UhpA在高渗应激下对基因表达调节的影响。
方法
(1)伤寒沙门菌uhpA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UhpA的基因缺陷变异株,根据伤寒沙门菌uhpA基因序列设计引物,扩增uhpA基因上、下游同源性片段,定向连接成uhpA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uhpA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 mM NaCl)LB培养液中分别培养伤寒沙门菌野生株和uhpA基因缺陷变异株4h(37℃,250r/min)至对数生长期,后转入高渗(300 mM NaCl)LB培养液中在同样条件下继续培养,在高渗应激30 min后,分别提取伤寒沙门菌野生株和uhpA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uhpA基因缺陷变异株在高渗应激下的基因表达谱差异。
(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。
(4)UhpA蛋白表达纯化:根据伤寒沙门菌uhpA基因序列设计引物,扩增uhpA基因片段,与表达载体pET-22b连接经热击法转化入大肠杆菌JM109,用IPTG诱导表达UhpA蛋白,用镍柱按产品说明纯化UhpA蛋白。
(5)凝胶阻滞试验:根据伤寒沙门菌cysD基因以及treB基因序列设计引物,设计特异性引物扩增出cysD基因以及treB基因启动子区域。取1-2ug PCR产物与不同浓度的UhpA蛋白混合,加入相应体积的GSM缓冲液,使三者反应总体积为20 ul,30℃孵育15 min。选取198 bp的真核生物PCR产物作为阴性对照,8%的丙烯酰胺胶电泳分离条带,溴化乙锭染色。
结果
(1)PCR及序列分析证实,uhpA基因缺陷变异株中uhpA基因297bp被6 bp取代,表明成功构建uhpA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uhpA基因缺陷变异株在高渗应激下有21个基因表达下调。绝大多数下调基因与硫代谢相关。
(3)对uhpA基因缺陷变异株中表达差异基因(cysM,treB),利用实时荧光定量PCR进行分析其mRNA水平,其结果与基因芯片分析结果基本一致。
(4)成功表达并纯化出UhpA蛋白,用于凝胶阻滞试验。
(5)选取uhpA基因缺陷变异株中表达差异基因(cysD,treB),凝胶阻滞试验的结果表明UhpA对treB,cysD等基因无直接调控作用。
结论
成功构建伤寒沙门菌uhpA基因缺陷变异株;UhpA在伤寒沙门菌高渗应激下对硫代谢基因表达调节发挥重要作用。
Objective
UhpA is a response regulator of two-component regulatory system UhpAB,and is highly expressed at hyperosmotic stress in Salmonella enterica serovar Typhi(S.Typhi).The goal of the study is to explore the infuluence of UhpA on gene expression of S.Typhi at 30 min of hyperosmotic stress.
Methods
1.Preparation of an uhpA deleted mutant of S.Typhi.The uhpA deleted mutant of S.Typhi was prepared by homologous recombination mediated by the suicide plasmid pGMB151.As the genomic information,two pairs of primers upper- and down-stream of the uhpA of S.Typhi were designed to amplify two homologous DNA fragments that were orientationally ligated to generate the homologous recombinant DNA fragment,which was then inserted into the suicide plasmid pGMB151 BamHⅠsite.The wild-type strain of S.Typhi was then transformed by the positive recombinant plasmid by electroporation.The uhpA deleted mutant was selected by screening on the LB with 5%sucrose plate and verified by PCR and DNA sequencing of the uhpA gene.
2.Transcriptional profile assay of S.Typhi.The environmental hyperosmotic stress was simulated by an osmotic up-shift,which increased the concentration of NaCl in the LB broth from 50 mM to 300 mM in vitro.Cells were grown in low osomtic LB(50 mM NaCl) (37℃,250 r/min) for 4 h to log-phase,subjected to an osomtic stress (increasing NaCl to 300 mM in LB),and then incubated for 30 min. The total RNA was extracted from the wild-type and the mutant uhpA~-of S.Typhi.cDNAs were synthesized by reverse transcription and labelling with cy3- or cy5-dCTP.The gene expression profiles of wild-type strain and uhpA mutant of S.Typhi at the osmotic stress after 30min were investigated by a geneomic DNA microarray analysis.
3.Quntitative real time PCR(qRT-PCR) analysis,qRT-PCR for some genes that have significant expressional difference in between wild-type and the uhpA mutant of S.Typhi was performed to prove the results of microarray analysis.
4.Expression and purification of UhpA.One pair of primers was designed to amplify DNA fragments of the uhpA gene of S.Typhi, which was then inserted into the expression plasmid pET-22b. Escherichia coli JM109 was then transformed by the expression plasmid by electroporation.Expression of the UhpA protein was induced by IPTG.UhpA protein was purified with Ni-colum according the manufactory instructions.
5.Gel-shifting experiment.Two pairs of primers were designed to amplify the promoter region of cysM and treB of S.Typhi.The mixtures containing 1-2μg PCR production and different amount of UhpA,GSM solution each 20μl were incubated at 30℃for 15 min befor electrophoresis.The PCR production of the eukaryotic cell was used as the negative control.The electrophoresis in 8%acrylamide gel was performed for separating PCR production.Gel was stained with Ethidium Bromide.
Results
1.PCR and sequencing analysis showed that the 297 bp of the uhpA ecoding region was deleted,suggesting that the uhpA gene deleted mutant of S.Typhi was constructed successfully.
2.Gene expression profiles analysis revealed that 21 genes in the uhpA mutant at hyperosmotic stress after 30 min were down-regulated.The most changed genes were associated with sulfur assimilation pathways.
3.Result of qRT-PCR showed that the expression difference of cysM and treB in between wild-type strain and the uhpA mutant was similar with the results from the microarray assay.
4.The UhpA-his_6 protein was expressed and purified successfully for gel-shifting experiments.
5.Result of gel-shifting experiments showed that UhpA could not bind poromoter regions of treB and cysM,sugesting that the expression of cysM and treB may be not regulated directly by the UhpA.
Conclusions
The uhpA gene deletion muatant of S.Typhi was constructed successfully. At up-shift high osmotic treatment,the UhpA may promoter the expression of genes involved in the metabolism of sulfur and trehalose indirectly.
UhpA为伤寒沙门菌双组份调节系统的效应调节蛋白,在高渗应激下其基因表达明显上调。本研究的目的是进一步探讨伤寒沙门菌调节因子UhpA在高渗应激下对基因表达调节的影响。
方法
(1)伤寒沙门菌uhpA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UhpA的基因缺陷变异株,根据伤寒沙门菌uhpA基因序列设计引物,扩增uhpA基因上、下游同源性片段,定向连接成uhpA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uhpA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 mM NaCl)LB培养液中分别培养伤寒沙门菌野生株和uhpA基因缺陷变异株4h(37℃,250r/min)至对数生长期,后转入高渗(300 mM NaCl)LB培养液中在同样条件下继续培养,在高渗应激30 min后,分别提取伤寒沙门菌野生株和uhpA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uhpA基因缺陷变异株在高渗应激下的基因表达谱差异。
(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。
(4)UhpA蛋白表达纯化:根据伤寒沙门菌uhpA基因序列设计引物,扩增uhpA基因片段,与表达载体pET-22b连接经热击法转化入大肠杆菌JM109,用IPTG诱导表达UhpA蛋白,用镍柱按产品说明纯化UhpA蛋白。
(5)凝胶阻滞试验:根据伤寒沙门菌cysD基因以及treB基因序列设计引物,设计特异性引物扩增出cysD基因以及treB基因启动子区域。取1-2ug PCR产物与不同浓度的UhpA蛋白混合,加入相应体积的GSM缓冲液,使三者反应总体积为20 ul,30℃孵育15 min。选取198 bp的真核生物PCR产物作为阴性对照,8%的丙烯酰胺胶电泳分离条带,溴化乙锭染色。
结果
(1)PCR及序列分析证实,uhpA基因缺陷变异株中uhpA基因297bp被6 bp取代,表明成功构建uhpA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uhpA基因缺陷变异株在高渗应激下有21个基因表达下调。绝大多数下调基因与硫代谢相关。
(3)对uhpA基因缺陷变异株中表达差异基因(cysM,treB),利用实时荧光定量PCR进行分析其mRNA水平,其结果与基因芯片分析结果基本一致。
(4)成功表达并纯化出UhpA蛋白,用于凝胶阻滞试验。
(5)选取uhpA基因缺陷变异株中表达差异基因(cysD,treB),凝胶阻滞试验的结果表明UhpA对treB,cysD等基因无直接调控作用。
结论
成功构建伤寒沙门菌uhpA基因缺陷变异株;UhpA在伤寒沙门菌高渗应激下对硫代谢基因表达调节发挥重要作用。
Objective
UhpA is a response regulator of two-component regulatory system UhpAB,and is highly expressed at hyperosmotic stress in Salmonella enterica serovar Typhi(S.Typhi).The goal of the study is to explore the infuluence of UhpA on gene expression of S.Typhi at 30 min of hyperosmotic stress.
Methods
1.Preparation of an uhpA deleted mutant of S.Typhi.The uhpA deleted mutant of S.Typhi was prepared by homologous recombination mediated by the suicide plasmid pGMB151.As the genomic information,two pairs of primers upper- and down-stream of the uhpA of S.Typhi were designed to amplify two homologous DNA fragments that were orientationally ligated to generate the homologous recombinant DNA fragment,which was then inserted into the suicide plasmid pGMB151 BamHⅠsite.The wild-type strain of S.Typhi was then transformed by the positive recombinant plasmid by electroporation.The uhpA deleted mutant was selected by screening on the LB with 5%sucrose plate and verified by PCR and DNA sequencing of the uhpA gene.
2.Transcriptional profile assay of S.Typhi.The environmental hyperosmotic stress was simulated by an osmotic up-shift,which increased the concentration of NaCl in the LB broth from 50 mM to 300 mM in vitro.Cells were grown in low osomtic LB(50 mM NaCl) (37℃,250 r/min) for 4 h to log-phase,subjected to an osomtic stress (increasing NaCl to 300 mM in LB),and then incubated for 30 min. The total RNA was extracted from the wild-type and the mutant uhpA~-of S.Typhi.cDNAs were synthesized by reverse transcription and labelling with cy3- or cy5-dCTP.The gene expression profiles of wild-type strain and uhpA mutant of S.Typhi at the osmotic stress after 30min were investigated by a geneomic DNA microarray analysis.
3.Quntitative real time PCR(qRT-PCR) analysis,qRT-PCR for some genes that have significant expressional difference in between wild-type and the uhpA mutant of S.Typhi was performed to prove the results of microarray analysis.
4.Expression and purification of UhpA.One pair of primers was designed to amplify DNA fragments of the uhpA gene of S.Typhi, which was then inserted into the expression plasmid pET-22b. Escherichia coli JM109 was then transformed by the expression plasmid by electroporation.Expression of the UhpA protein was induced by IPTG.UhpA protein was purified with Ni-colum according the manufactory instructions.
5.Gel-shifting experiment.Two pairs of primers were designed to amplify the promoter region of cysM and treB of S.Typhi.The mixtures containing 1-2μg PCR production and different amount of UhpA,GSM solution each 20μl were incubated at 30℃for 15 min befor electrophoresis.The PCR production of the eukaryotic cell was used as the negative control.The electrophoresis in 8%acrylamide gel was performed for separating PCR production.Gel was stained with Ethidium Bromide.
Results
1.PCR and sequencing analysis showed that the 297 bp of the uhpA ecoding region was deleted,suggesting that the uhpA gene deleted mutant of S.Typhi was constructed successfully.
2.Gene expression profiles analysis revealed that 21 genes in the uhpA mutant at hyperosmotic stress after 30 min were down-regulated.The most changed genes were associated with sulfur assimilation pathways.
3.Result of qRT-PCR showed that the expression difference of cysM and treB in between wild-type strain and the uhpA mutant was similar with the results from the microarray assay.
4.The UhpA-his_6 protein was expressed and purified successfully for gel-shifting experiments.
5.Result of gel-shifting experiments showed that UhpA could not bind poromoter regions of treB and cysM,sugesting that the expression of cysM and treB may be not regulated directly by the UhpA.
Conclusions
The uhpA gene deletion muatant of S.Typhi was constructed successfully. At up-shift high osmotic treatment,the UhpA may promoter the expression of genes involved in the metabolism of sulfur and trehalose indirectly.
引文
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