大肠杆菌应激诱导基因表达谱芯片分析
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摘要
应激是影响微生物发酵生产的重要因素。为挖掘细菌耐热和化合物胁迫等调控相关基因,揭示微生物应激反应调控的分子机制,采用Affymetrix全基因组表达谱芯片,对42℃热激和0.1 mol/L CaCl2处理的大肠杆菌E. coli基因谱进行分析。结果表明,应激处理菌体中共有583个差异表达基因,包括374个表达量上调和209个表达量下调差异基因;GO功能分类将差异表达基因分为34类,大部分差异基因的分子功能覆盖结合、催化和转运等生物学活性;差异基因KEGG分析显示富集在28个通路,以ABC转运蛋白、鞭毛组装、双组分系统、尿素循环和氨基酸新陈代谢以及Ⅲ型分泌系统等通路为主要代表;应激胁迫的E. coli主要通过噬菌体休克蛋白操纵子(psp)基因进行逆境适应性转录调节,同时伴随着酶和氨基酸的合成基因以及细胞氧化还原态平衡基因的调控变化。
Stress is an important factor for affecting the microorganism fermentation production. In order to find the regulation genes related to high temperature and compound resistant, and for further reveal the molecular mechanism of regulation of microbial stress response, the genic spectrum of Escherichia coli treated by 42℃heat shock and 0.1 mol/L CaCl2 were analyzed using the whole genome microarray Affymetrix. Results showed that 583 differentially expressed genes were detected in Escherichia coli treated with two kinds stresses,including 374 up-regulated genes and 209 down-regulated genes. These genes could be divided into 34 categories according to the GO functional classification, whose molecular function most involved in binding,catalysis and transport activities. KEGG analysis suggested that these differential genes were enriched in 28 pathways, with ABC transporters, flagellum assembly, two-component system, urea cycle and amino acid metabolism, as well as Ⅲ secretion system as the main representative. Escherichia coli under stress mainly regulated stress adaptive transcription through the phage shock protein operon(psp) genes. At the same time,the synthesis of enzymes and amino acids and regulation of oxidative reduction equilibrium gene were also involved.
Stress is an important factor for affecting the microorganism fermentation production. In order to find the regulation genes related to high temperature and compound resistant, and for further reveal the molecular mechanism of regulation of microbial stress response, the genic spectrum of Escherichia coli treated by 42℃heat shock and 0.1 mol/L CaCl2 were analyzed using the whole genome microarray Affymetrix. Results showed that 583 differentially expressed genes were detected in Escherichia coli treated with two kinds stresses,including 374 up-regulated genes and 209 down-regulated genes. These genes could be divided into 34 categories according to the GO functional classification, whose molecular function most involved in binding,catalysis and transport activities. KEGG analysis suggested that these differential genes were enriched in 28 pathways, with ABC transporters, flagellum assembly, two-component system, urea cycle and amino acid metabolism, as well as Ⅲ secretion system as the main representative. Escherichia coli under stress mainly regulated stress adaptive transcription through the phage shock protein operon(psp) genes. At the same time,the synthesis of enzymes and amino acids and regulation of oxidative reduction equilibrium gene were also involved.
引文
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Dong C.,Cao J.,Zhang J.,and Shen B.,2008,High expression of thermostable alpha amylase gene in Bacillus subtilis,Yingy ong Yu Huanjing Shengwuxuebao(Chinese Journal of Applied and Environmental Biology),(4):534-538(董晨,曹娟,张迹,沈标,2008,耐高温α-淀粉酶基因在枯草芽孢杆菌中的高效表达,应用与环境生物学报,(4):534-538)
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Hao Y.H.,Zhang W.,and Chen M.,2012,Research progress of bacterial two-component system,Zhongguo Nongyekeji Daobao(Journal of Agricultural Science and Technoligy),14(2):67-72(郝艳华,张维,陈明,2012,细菌双组分系统的研究进展,中国农业科技导报,14(2):67-72)
Hirota Y.,Yasuda S.,Yamada M.,Nishimura A.,SugimotoK.,Sugisaki H.,and Takanami M.,1979,Structural and functional properties of the Escherichia coli origin of DNA replication,Cold Spring Harb Symp Quant Biol.,43 Pt 1:129-138
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Liu Y.P.,Tang H.Z.,and Xu P.,2014,Research progress on improving microbial resistance to stress,Guangzhou Huagong(Guangzhou Chemical Industry),(24):4-6(刘玉萍,唐鸿志,许平,2014,提高微生物抗逆性技术的研究进展,广州化工,(24):4-6)
Li Z.H.,2008,Research progress in typeⅢsecretion system,Yixue Xinxi(Medical Information),(11):2119-2122(李正花,2008,%Ⅲ型分泌系统的研究进展,医学信息,(11):2119-2122)
Lloyd L.J.,Jones S.E.,Jovanovic G.,Gyaneshwar P.,Rolfe M.D.,Thompson A.,Hinton J.C.,and Buck M.,2004,Identification of a new member of the phage shock protein response in Escherichia coli,the phage shock protein G(PspG),J.Biol.Chem.,279(53):55707-55714
Martin J.E.,Waters L.S.,Storz G.,and Imlay J.A.,2015,The Escherichia coli small protein Mnt S and exporter MntP optimize the intracellular concentration of manganese,PLo SGenet,11(3):e1004977
Newman D.J.,and Cragg G.M.,2012,Natural products as sources of new drugs over the 30 years from 1981 to 2010,Journal of Natural Products,75(3):311-335
Peltoniemi M.J.,Karala A.R.,Jurvansuu J.K.,Kinnula V.L.,and Ruddock L.W.,2006,Insights into deglutathionylation reactions different intermediates in the glutaredoxin and protein disulfide isomerase catalyzed reactions are defined by the gamma-linkage present in glutathione,J.Biol.Chem.,281(44):33107-33114
Saaranen M.J.,Salo K.E.,Latva-Ranta M.K.,Kinnula V.L.,and Ruddock L.W.,2009,The C-terminal active site cysteine of Escherichia coli glutaredoxin 1 determines the glutathione specificity of the second step of peptide deglutathionylation,Antioxid Redox Signal,11(8):1819-1828
Sambrook J.,and Russell D.W.,eds.,Huang P.T.,Wang J.X.,and Zhu H.C.,trans.,2002,Third Edition of experimental guide on Molecular clone,Beijing Science Press,Beijing,China,pp.96-99(萨姆布鲁克J.,拉塞尔D.W.,著,黄培堂,王嘉玺,朱厚础,译,2002,分子克隆实验指南第三版,北京科学出版社,中国,北京,pp.96-99)
Schena M.,Shalon D.,Davis R.W.,and Brown P.O.,1995,Quantitative monitoring of gene expression patterns with a complementary DNA microarray,Science,270:467-470
Sun J.,2015,Rhine chlamydomonas intraflagellar transport protein IFT81 regulates flagellar assembly,Gongchengkexue Xuebao(Chinese Journal of Engineering),37(8):1105-1109(孙娟,2015,莱茵衣藻鞭毛内运输蛋白IFT81调控鞭毛组装,工程科学学报,37(8):1105-1109)
Wang F.J.,Lu F.,Zhang J.,and Xue W.T.,2007,Research progress of soybean dregs fermention food by microorganism,Shipin Gongye Keji(Science and Technology of Food Industry),(9):212-215(王夫杰,鲁绯,张建,薛文通,2007,微生物发酵豆渣食品的研究进展,食品工业科技,(9):212-215)
Wang J.,Shi Y.C.,and Zhang J.,2012,Advances in studies on anti adversity genes derived from microorganisms,Gaoshi Like Xuekan(Journal of Science Teachers College and University),32(1):59-62(王婧,石运臣,张建,2012,源于微生物的抗逆基因研究进展,高师理科学刊,32(1):59-62)
Wang L.Q.,and Tan H.R.,2009,Molecular regulation of microbial secondary metabolism,Weishengwu Xuebao(Acta Microbiologica Sinica),(4):411-416(王琳淇,谭华荣,2009,微生物次生代谢的分子调控,微生物学报,(4):411-416)
Yao L.,Fu L.Y.,Wang Y.Q.,Yuan C.B.,Yang G.,and Luo J.F.,2016,Orthogonal experiment and gas production potention on factors influencing anaerobic fermentation of reed stalk,Huanjinggongcheng Xuebao(Chinese Journal of Environmental engineering),(11):6692-6698(姚利,付龙云,王艳芹,袁长波,杨光,罗加法,2016,芦苇秸秆厌氧发酵影响因素正交实验及产气潜力研究,环境工程学报,(11):6692-6698)
Ye J.,Fang L.,Zheng H.,Zhang Y.,Chen J.,Zhang Z.,Wang J.,Li S.,Li R.,Bolund L.,and Wang J.,2006,WEGO:a web tool for plotting GO annotations,Nucleic Acids Research,34:293-297
Yunes R.A.,Yunes E.U.,Poluektova M.S.,Dyachkova K.M.,Klimina A.S.,Kovtun O.V.,Averina V.S.,Orlova V.N.,and Danilenko,2016,GABA production and structure of gadB/gadC genes in Lactobacillus and Bifidobacterium strains from human microbiota,Anaerobe,42:197-204
Zhang W.Z.,and Guo J.H.,2013,Research progress in optimization of microbial fermentation technology,Guangdong nongye Kexue(Guangdong Agricultural Sciences),(6):114-117(张文芝,郭坚华,2013,微生物发酵工艺优化研究进展,广东农业科学,(6):114-117)
Dai X.Y.,Wang Y.Q.,Zhou J.,Yang B.,and Wang L.Q.,2000,Metabolic regulation of trehalose in Escherichia coli,Shenwu Gongcheng Jinzhan(Progress in Biotechnology),(6):26-29(戴秀玉,王忆琴,周坚,杨波,王连琴,2000,大肠杆菌海藻糖的代谢调控,生物工程进展,(6):26-29)
Dean M.,Rzhetsky A.,and Allikmets R.,2001,The human ATP-binding cassette(ABC)transporter superfamily,Genome Res.,11(7):1156-1166
Dong C.,Cao J.,Zhang J.,and Shen B.,2008,High expression of thermostable alpha amylase gene in Bacillus subtilis,Yingy ong Yu Huanjing Shengwuxuebao(Chinese Journal of Applied and Environmental Biology),(4):534-538(董晨,曹娟,张迹,沈标,2008,耐高温α-淀粉酶基因在枯草芽孢杆菌中的高效表达,应用与环境生物学报,(4):534-538)
Farnham P.J.,and Platt T.,1982,Effects of DNA base analogs on transcription termination at the tryptophan operon attenuator of Escherichia coli,Proc.Natl.Acad.Sci.USA,79(4):998-1002
Hao Y.H.,Zhang W.,and Chen M.,2012,Research progress of bacterial two-component system,Zhongguo Nongyekeji Daobao(Journal of Agricultural Science and Technoligy),14(2):67-72(郝艳华,张维,陈明,2012,细菌双组分系统的研究进展,中国农业科技导报,14(2):67-72)
Hirota Y.,Yasuda S.,Yamada M.,Nishimura A.,SugimotoK.,Sugisaki H.,and Takanami M.,1979,Structural and functional properties of the Escherichia coli origin of DNA replication,Cold Spring Harb Symp Quant Biol.,43 Pt 1:129-138
Li M.,2003,Anaerobic respiration and fermentation of microorganisms and their application in production and environmen tal protection,Shengwuxue Tongbao(Bulletin of Biology),(5):20-22(李明,2003,微生物的无氧呼吸、发酵及其在生产和环境保护上的应用,生物学通报,(5):20-22)
Liu Y.P.,Tang H.Z.,and Xu P.,2014,Research progress on improving microbial resistance to stress,Guangzhou Huagong(Guangzhou Chemical Industry),(24):4-6(刘玉萍,唐鸿志,许平,2014,提高微生物抗逆性技术的研究进展,广州化工,(24):4-6)
Li Z.H.,2008,Research progress in typeⅢsecretion system,Yixue Xinxi(Medical Information),(11):2119-2122(李正花,2008,%Ⅲ型分泌系统的研究进展,医学信息,(11):2119-2122)
Lloyd L.J.,Jones S.E.,Jovanovic G.,Gyaneshwar P.,Rolfe M.D.,Thompson A.,Hinton J.C.,and Buck M.,2004,Identification of a new member of the phage shock protein response in Escherichia coli,the phage shock protein G(PspG),J.Biol.Chem.,279(53):55707-55714
Martin J.E.,Waters L.S.,Storz G.,and Imlay J.A.,2015,The Escherichia coli small protein Mnt S and exporter MntP optimize the intracellular concentration of manganese,PLo SGenet,11(3):e1004977
Newman D.J.,and Cragg G.M.,2012,Natural products as sources of new drugs over the 30 years from 1981 to 2010,Journal of Natural Products,75(3):311-335
Peltoniemi M.J.,Karala A.R.,Jurvansuu J.K.,Kinnula V.L.,and Ruddock L.W.,2006,Insights into deglutathionylation reactions different intermediates in the glutaredoxin and protein disulfide isomerase catalyzed reactions are defined by the gamma-linkage present in glutathione,J.Biol.Chem.,281(44):33107-33114
Saaranen M.J.,Salo K.E.,Latva-Ranta M.K.,Kinnula V.L.,and Ruddock L.W.,2009,The C-terminal active site cysteine of Escherichia coli glutaredoxin 1 determines the glutathione specificity of the second step of peptide deglutathionylation,Antioxid Redox Signal,11(8):1819-1828
Sambrook J.,and Russell D.W.,eds.,Huang P.T.,Wang J.X.,and Zhu H.C.,trans.,2002,Third Edition of experimental guide on Molecular clone,Beijing Science Press,Beijing,China,pp.96-99(萨姆布鲁克J.,拉塞尔D.W.,著,黄培堂,王嘉玺,朱厚础,译,2002,分子克隆实验指南第三版,北京科学出版社,中国,北京,pp.96-99)
Schena M.,Shalon D.,Davis R.W.,and Brown P.O.,1995,Quantitative monitoring of gene expression patterns with a complementary DNA microarray,Science,270:467-470
Sun J.,2015,Rhine chlamydomonas intraflagellar transport protein IFT81 regulates flagellar assembly,Gongchengkexue Xuebao(Chinese Journal of Engineering),37(8):1105-1109(孙娟,2015,莱茵衣藻鞭毛内运输蛋白IFT81调控鞭毛组装,工程科学学报,37(8):1105-1109)
Wang F.J.,Lu F.,Zhang J.,and Xue W.T.,2007,Research progress of soybean dregs fermention food by microorganism,Shipin Gongye Keji(Science and Technology of Food Industry),(9):212-215(王夫杰,鲁绯,张建,薛文通,2007,微生物发酵豆渣食品的研究进展,食品工业科技,(9):212-215)
Wang J.,Shi Y.C.,and Zhang J.,2012,Advances in studies on anti adversity genes derived from microorganisms,Gaoshi Like Xuekan(Journal of Science Teachers College and University),32(1):59-62(王婧,石运臣,张建,2012,源于微生物的抗逆基因研究进展,高师理科学刊,32(1):59-62)
Wang L.Q.,and Tan H.R.,2009,Molecular regulation of microbial secondary metabolism,Weishengwu Xuebao(Acta Microbiologica Sinica),(4):411-416(王琳淇,谭华荣,2009,微生物次生代谢的分子调控,微生物学报,(4):411-416)
Yao L.,Fu L.Y.,Wang Y.Q.,Yuan C.B.,Yang G.,and Luo J.F.,2016,Orthogonal experiment and gas production potention on factors influencing anaerobic fermentation of reed stalk,Huanjinggongcheng Xuebao(Chinese Journal of Environmental engineering),(11):6692-6698(姚利,付龙云,王艳芹,袁长波,杨光,罗加法,2016,芦苇秸秆厌氧发酵影响因素正交实验及产气潜力研究,环境工程学报,(11):6692-6698)
Ye J.,Fang L.,Zheng H.,Zhang Y.,Chen J.,Zhang Z.,Wang J.,Li S.,Li R.,Bolund L.,and Wang J.,2006,WEGO:a web tool for plotting GO annotations,Nucleic Acids Research,34:293-297
Yunes R.A.,Yunes E.U.,Poluektova M.S.,Dyachkova K.M.,Klimina A.S.,Kovtun O.V.,Averina V.S.,Orlova V.N.,and Danilenko,2016,GABA production and structure of gadB/gadC genes in Lactobacillus and Bifidobacterium strains from human microbiota,Anaerobe,42:197-204
Zhang W.Z.,and Guo J.H.,2013,Research progress in optimization of microbial fermentation technology,Guangdong nongye Kexue(Guangdong Agricultural Sciences),(6):114-117(张文芝,郭坚华,2013,微生物发酵工艺优化研究进展,广东农业科学,(6):114-117)