伤寒沙门菌mig-14基因功能研究
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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。
Objective
Previous 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.
Methods
The 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.
Results
PCR 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.
Conclusions
The 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.
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。
Objective
Previous 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.
Methods
The 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.
Results
PCR 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.
Conclusions
The 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.
引文
1.Christopher M,Parry MB,Hien T,et al.Review Article:Typhoid fever.N Engl Med,2002,347:1770-1781
2.Everest P,Wain J,Roberts M,et al.The molecular mechanisms of severe typhoid fever.Trends Microbiol,2001,9:316-320
3.House D,Bishop A,Parry C,et al.Typhoid fever:pathogenesis and disease.Curr Opin Infect Dis,2001,14:573-578
4.Jones BD,Falkow S.Salmonellosis:host immune responses and bacterial virulence determinants.Annu Rev Immunol,1996,14:533-5361
5.Darwin KH,Mille VL.Molecular basis of the interaction of Salmonella with the intestinal mucosa.Clin Microbiol Rev,1999,12:405-428
6.Leclerc GJ,Tartera C,Metcalf ES.Environmental regulation of Salmonella typhi invasion-defective mutants.Infect Immun,1998,66:682-691
7.Tartera C,Metcalf ES.Osmolarity and growth phase overlap in regulation of Salmonella typhi adherence to and invasion of human intestinal cells.Infect Immun,1993,61:3084-3089
8.Zhao L,Ezaki T,Li ZY,et al.Vi-Suppressed wild strain Salmonella typhi cultured in high osmolarity is hyperinvasive toward epithelial cells and destructive of Peyer's patches.Microbiol Immunol,2001,45:149-158
9.Valdivia RH,Falkow S.Fluorescence-based isolation of bacterial genes expressed within host cells.Science,1997,277:2007-2011
10.Valdivia RH,Cirillo DM,Lee AK,et al.mig-14 is a horizontally acquired,host-induced gene required for Salmonella enterica lethal infection in the murine model of typhoid fever.Infect Immun,2000:7126-7131
11.Brodsky IE,Robert KE,Miller SI,et al.mig-14 is a Salmonella gene that plays a role in bacterial resistance to antimicrobial peptides.J Bacteriol,2002,184:3203-3213
12.Navarre WW,Halsey TA,Walthers D,et al.Co-regulation of Salmonella enterica genes required for virulence and resistance to antimicrobial peptides by SlyA and PhoP/PhoQ.Mol Microbiol,2005,56:492-508
13.Huang XX,Xu HX,Sun XS,et al.Genome-Wide scan of the gene expression kinetics of Salmonella enterica serovar Typhi during hyperosmotic stress.Int J Mol Sci,2007,8:116-135
14.Zasloff M.Antibiotic peptides as mediators of innate immunity.Curr Opin Immunol,1992:3-7
15.Zasloff M.Antimicrobial peptides of multicellular organisms.Nature,2002,415:389-395.
16.Trent MS.Biosynthesis,transport,and modification of lipid A.Biochem Cell Biol,2004,82:71-86
17.Gunn JS,Lim KB,Krueger J,et al.PmrA-PmrB regulated genes necessary for 4-aminoarabinose lipid A modification and polymyxin resistance.Mol Microbiol,1998:1171-1182
18.Lee H,Hsu F,Turk J,et al.The pmrA-regulated pmrC gene mediates phosphoethanolamine modification of lipid A and polymyxin resistance in Salmonella enterica.J Bacteriol,2004,279:4124-4133
19.Nishino K,Hsu F,Turk J,et al.Identification of the lipopolysaccharide modifications controlled by the Salmonella PmrA/PmrB system mediating resistance to Fe(Ⅲ) and Al(Ⅲ).Mol Microbiol,2006,61:645-654
20.Shi Y,Cromie MJ,Hsu F,et al.PhoP-regulated Salmonella resistance to the antimicrobial peptides magainin 2 and polymyxin B.Mol Microbiol,2004,53:229-241
21.Zhou Z,Ribeiro AA,Lin S,et al.Lipid A modifications in polymyxin-resistant Salmonella typhimurium.J Biol Chem,2001,276:43111-43121
22.Breazeale SD,Ribeiro AA,Raetz CR.Origin of lipid A species modified with 4-amino-4-deoxy-L-arabinose in polymyxin-resistant mutants of Escherichia coli.J Biol Chem,2003,278,24731-24739
23.Shi Y,Latifi T,Cromie MJ,et al.Transcriptional control of the antimicrobial peptide resistance ugtL gene by the Salmonella PhoP and SlyA regulatory proteins.J Bacteriol,2004,279:38618-38625
24.Detweiler CS,Monack DM,Brodsky IE,et al.virK,somA and rcsC are important for systemic Salmonella enterica serovar Typhimurium infection and cationic peptide resistance.Mol Microbiol,2003,48:385-400
25.Costerton JW,Lewandowski Z,Caldwell DE,et al.Microbial bio films.Annu Rev Microbio,1995,115:711-745
26.Mah TF,O'Toole GA.Mechanisms of biofilm resistance to antimicrobial agents.Trends Microbiol,2001,9:34-39
27.Spiers AJ,Rainey PB.The Pseudomonas Xuorescens SBW25 wrinkly spreader biofilm requires attachment factor,cellulose fibre and LPS interactions to maintain strength and integrity.Microbiol,2005,151:2829-2839
28.Brodsky IE,Ghori N,Falkow S,et al.Mig-14 is an inner membrane-associated protein that promotes Salmonella typhimurium resistance to CRAMP,survival within activated macrophages and persistent infection.Mol Microbiol,2005,55:954-972
29.Kaniga K,Delor I,Comelis GR.A wide-host-range suicide vector for imp roving reverse genetics in gram negative bacteria:inactivation of the blaA gene of Yersinia enterocolitica.Gene,1991,109:137-141
30.生秀梅,黄新祥,茅凌翔等.伤寒沙门菌基因组DNA芯片的制备与基因表达谱分析应用.生物化学与生物物理进展.2009,36:206-212
31.Talat AM,Hunter P,Johnston SA.Genome-directed primers for selective labeling of bacterial transcripts for DNA microarray analysis.Nat Biotechnol,2000,18:679-682
32.周冬生,杨瑞馥,宋亚军,等.DNA芯片技术在微生物研究中的应用.军事医学科学院院刊.2002,26:294-300
33.Chilcott GS,Hughes KT.Coupling of flagellar gene expression to flagellar assembly in Salmonella enterica serovar Typhimurium and Escherichia coli.Microbil Mol Biol Rev,2000,64:694-708
34.Bishop RE,Gibbons HS,Guina T,et al.Transfer ofpalmitate from phospholipids to lipid A in outer membranes of Gram-negative bacteria.EMBO J,2000,19:5071-5080
35.Guo L,Lim KB,Poduje CM,et al.Lipid A acylation and bacterial resistance against vertebrate antimicrobial peptides.Cell,1998,95:189-198
36.Duong T,Barrangou R,Russell WM,et al.Characterization of the tre locus and analysis of trehalose cryoprotection in Lactobacillus acidophilus NCFM.Appl Environ Microbiol,2006,72:1218-1225
37.Lee ML,Kuo Fc,Whimore GA,et al.Importance of replication in microarray gene expression studies:statistical methods and evidence from repetitive cDNA hybirdizations.Proc Natl Acad Sci,2000,97:9834-9839
38.Lekanne Deprez RH,Fijnvandraat AC,Ruijter JM,et al.Sensitivity and accuracy of quantitative real-time polymerase chain reaction using SYBR green Ⅰ depends on cDNA synthesis conditions.Anal Biochem,2002,307:63-69
39.Annapula G,Lut O.An overview of real-time PCR:application to quantify cytokine gene expression.Method,2001,25:386-401
2.Everest P,Wain J,Roberts M,et al.The molecular mechanisms of severe typhoid fever.Trends Microbiol,2001,9:316-320
3.House D,Bishop A,Parry C,et al.Typhoid fever:pathogenesis and disease.Curr Opin Infect Dis,2001,14:573-578
4.Jones BD,Falkow S.Salmonellosis:host immune responses and bacterial virulence determinants.Annu Rev Immunol,1996,14:533-5361
5.Darwin KH,Mille VL.Molecular basis of the interaction of Salmonella with the intestinal mucosa.Clin Microbiol Rev,1999,12:405-428
6.Leclerc GJ,Tartera C,Metcalf ES.Environmental regulation of Salmonella typhi invasion-defective mutants.Infect Immun,1998,66:682-691
7.Tartera C,Metcalf ES.Osmolarity and growth phase overlap in regulation of Salmonella typhi adherence to and invasion of human intestinal cells.Infect Immun,1993,61:3084-3089
8.Zhao L,Ezaki T,Li ZY,et al.Vi-Suppressed wild strain Salmonella typhi cultured in high osmolarity is hyperinvasive toward epithelial cells and destructive of Peyer's patches.Microbiol Immunol,2001,45:149-158
9.Valdivia RH,Falkow S.Fluorescence-based isolation of bacterial genes expressed within host cells.Science,1997,277:2007-2011
10.Valdivia RH,Cirillo DM,Lee AK,et al.mig-14 is a horizontally acquired,host-induced gene required for Salmonella enterica lethal infection in the murine model of typhoid fever.Infect Immun,2000:7126-7131
11.Brodsky IE,Robert KE,Miller SI,et al.mig-14 is a Salmonella gene that plays a role in bacterial resistance to antimicrobial peptides.J Bacteriol,2002,184:3203-3213
12.Navarre WW,Halsey TA,Walthers D,et al.Co-regulation of Salmonella enterica genes required for virulence and resistance to antimicrobial peptides by SlyA and PhoP/PhoQ.Mol Microbiol,2005,56:492-508
13.Huang XX,Xu HX,Sun XS,et al.Genome-Wide scan of the gene expression kinetics of Salmonella enterica serovar Typhi during hyperosmotic stress.Int J Mol Sci,2007,8:116-135
14.Zasloff M.Antibiotic peptides as mediators of innate immunity.Curr Opin Immunol,1992:3-7
15.Zasloff M.Antimicrobial peptides of multicellular organisms.Nature,2002,415:389-395.
16.Trent MS.Biosynthesis,transport,and modification of lipid A.Biochem Cell Biol,2004,82:71-86
17.Gunn JS,Lim KB,Krueger J,et al.PmrA-PmrB regulated genes necessary for 4-aminoarabinose lipid A modification and polymyxin resistance.Mol Microbiol,1998:1171-1182
18.Lee H,Hsu F,Turk J,et al.The pmrA-regulated pmrC gene mediates phosphoethanolamine modification of lipid A and polymyxin resistance in Salmonella enterica.J Bacteriol,2004,279:4124-4133
19.Nishino K,Hsu F,Turk J,et al.Identification of the lipopolysaccharide modifications controlled by the Salmonella PmrA/PmrB system mediating resistance to Fe(Ⅲ) and Al(Ⅲ).Mol Microbiol,2006,61:645-654
20.Shi Y,Cromie MJ,Hsu F,et al.PhoP-regulated Salmonella resistance to the antimicrobial peptides magainin 2 and polymyxin B.Mol Microbiol,2004,53:229-241
21.Zhou Z,Ribeiro AA,Lin S,et al.Lipid A modifications in polymyxin-resistant Salmonella typhimurium.J Biol Chem,2001,276:43111-43121
22.Breazeale SD,Ribeiro AA,Raetz CR.Origin of lipid A species modified with 4-amino-4-deoxy-L-arabinose in polymyxin-resistant mutants of Escherichia coli.J Biol Chem,2003,278,24731-24739
23.Shi Y,Latifi T,Cromie MJ,et al.Transcriptional control of the antimicrobial peptide resistance ugtL gene by the Salmonella PhoP and SlyA regulatory proteins.J Bacteriol,2004,279:38618-38625
24.Detweiler CS,Monack DM,Brodsky IE,et al.virK,somA and rcsC are important for systemic Salmonella enterica serovar Typhimurium infection and cationic peptide resistance.Mol Microbiol,2003,48:385-400
25.Costerton JW,Lewandowski Z,Caldwell DE,et al.Microbial bio films.Annu Rev Microbio,1995,115:711-745
26.Mah TF,O'Toole GA.Mechanisms of biofilm resistance to antimicrobial agents.Trends Microbiol,2001,9:34-39
27.Spiers AJ,Rainey PB.The Pseudomonas Xuorescens SBW25 wrinkly spreader biofilm requires attachment factor,cellulose fibre and LPS interactions to maintain strength and integrity.Microbiol,2005,151:2829-2839
28.Brodsky IE,Ghori N,Falkow S,et al.Mig-14 is an inner membrane-associated protein that promotes Salmonella typhimurium resistance to CRAMP,survival within activated macrophages and persistent infection.Mol Microbiol,2005,55:954-972
29.Kaniga K,Delor I,Comelis GR.A wide-host-range suicide vector for imp roving reverse genetics in gram negative bacteria:inactivation of the blaA gene of Yersinia enterocolitica.Gene,1991,109:137-141
30.生秀梅,黄新祥,茅凌翔等.伤寒沙门菌基因组DNA芯片的制备与基因表达谱分析应用.生物化学与生物物理进展.2009,36:206-212
31.Talat AM,Hunter P,Johnston SA.Genome-directed primers for selective labeling of bacterial transcripts for DNA microarray analysis.Nat Biotechnol,2000,18:679-682
32.周冬生,杨瑞馥,宋亚军,等.DNA芯片技术在微生物研究中的应用.军事医学科学院院刊.2002,26:294-300
33.Chilcott GS,Hughes KT.Coupling of flagellar gene expression to flagellar assembly in Salmonella enterica serovar Typhimurium and Escherichia coli.Microbil Mol Biol Rev,2000,64:694-708
34.Bishop RE,Gibbons HS,Guina T,et al.Transfer ofpalmitate from phospholipids to lipid A in outer membranes of Gram-negative bacteria.EMBO J,2000,19:5071-5080
35.Guo L,Lim KB,Poduje CM,et al.Lipid A acylation and bacterial resistance against vertebrate antimicrobial peptides.Cell,1998,95:189-198
36.Duong T,Barrangou R,Russell WM,et al.Characterization of the tre locus and analysis of trehalose cryoprotection in Lactobacillus acidophilus NCFM.Appl Environ Microbiol,2006,72:1218-1225
37.Lee ML,Kuo Fc,Whimore GA,et al.Importance of replication in microarray gene expression studies:statistical methods and evidence from repetitive cDNA hybirdizations.Proc Natl Acad Sci,2000,97:9834-9839
38.Lekanne Deprez RH,Fijnvandraat AC,Ruijter JM,et al.Sensitivity and accuracy of quantitative real-time polymerase chain reaction using SYBR green Ⅰ depends on cDNA synthesis conditions.Anal Biochem,2002,307:63-69
39.Annapula G,Lut O.An overview of real-time PCR:application to quantify cytokine gene expression.Method,2001,25:386-401