Red两步同源重组法在大肠杆菌基因敲除中的应用
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摘要
基因敲除是研究基因功能最直接的方法。对于大肠杆菌(Escherichia coli,E.coli)而言,传统的基因敲除方法是利用其原有的RecA系统,此方法依赖于特定的限制性内切酶酶切位点,且所需同源臂长,操作复杂。Red同源重组法的出现使得基因组的改造更为快速、简单,在E.coli基因敲除中的应用也愈加广泛和成熟。作者介绍了Red同源重组系统的组成和同源重组原理,阐述了常用的两步同源重组法敲除E.coli基因的操作步骤及注意事项。大肠杆菌的两步法基因敲除技术已在工程菌改造、病原菌致病机理、耐药机制研究等领域做出巨大贡献,由于两步法仍保留有Red同源重组系统电转效率低、有序列残留的缺陷,作者对几个针对两步法的经典的无痕化改造进行了总结介绍,通过对以上方法及应用进行综述,为以大肠杆菌作为工程菌的基因敲除技术提供参考。
Knock-out is the most direct way to explore gene's function.The traditional method of Escherichia coli(E.coli)gene knock-out is to use its own RecA reorganization system,which relies on the specific given cleavage sites of restriction enzymes,requires long homologous arm,furthermore,the operation is complex.Modification of the genome has become simple and rapid since the advent of Red homologous recombination method.And its application in E.coli gene knock-out has been more mature.The structure and functional principle of Red homologous recombination is introduced,besides,this review expounds the operating steps and notices of the common two-step Red-mediated recombination.The Red homologous recombination technology in E.coli has made great contribution in the area of modifications of engineering strain,pathogenicity and bacterial resistance of the pathogenic bacteria.Additionally,the advantages and shortcomings of the method are set forth.According to the disadvantage that the traditional method do leave recombinase recognition site scars and has low efficiency,a sum of classical methods for scar-less gene replacement are described in the final part of the article.These approach can provide better applications for the construction of E.coli genome.
Knock-out is the most direct way to explore gene's function.The traditional method of Escherichia coli(E.coli)gene knock-out is to use its own RecA reorganization system,which relies on the specific given cleavage sites of restriction enzymes,requires long homologous arm,furthermore,the operation is complex.Modification of the genome has become simple and rapid since the advent of Red homologous recombination method.And its application in E.coli gene knock-out has been more mature.The structure and functional principle of Red homologous recombination is introduced,besides,this review expounds the operating steps and notices of the common two-step Red-mediated recombination.The Red homologous recombination technology in E.coli has made great contribution in the area of modifications of engineering strain,pathogenicity and bacterial resistance of the pathogenic bacteria.Additionally,the advantages and shortcomings of the method are set forth.According to the disadvantage that the traditional method do leave recombinase recognition site scars and has low efficiency,a sum of classical methods for scar-less gene replacement are described in the final part of the article.These approach can provide better applications for the construction of E.coli genome.
引文
[1]阳燕,杨英明,胡涛.原核微生物基因敲除策略的研究进展[J].国际口腔科学杂志,2016,43(5):578-583.
[2]Wang S.DotU expression is highly induced during in vivoinfection and responsible for virulence and Hcp1secretion in avian pathogenic Escherichia coli[J].Frontiers in Microbiology,2014,5:588.
[3]Wang S.IbeR facilitates stress-resistance,invasion and pathogenicity of avian pathogenic Escherichia coli[J].PLoS One,2015,10(3):e0119698.
[4]李继东,才学鹏.山羊痘病毒TK基因功能缺失对其增殖的影响[J].中国畜牧兽医,2017,44(2):319-326.
[5]赵雪丽,闫若潜,吴志明,等.鉴别猪伪狂犬病病毒gE基因缺失疫苗和野毒感染的二重PCR诊断方法的建立和应用[J].中国畜牧兽医,2015,42(4):865-870.
[6]陈柳,余斌,倪征,等.表达小鹅瘟病毒VP2蛋白重组鸭瘟病毒的构建及其生物学特性[J].中国农业科学,2016,49(14):2813-2821.
[7]周化民,孙旭利,陈建明.同源重组和重组酶Rad51的调控[J].生物化学与生物物理进展,2016,12:1154-1162.
[8]卫亚红,段敏,向照举,等.降解布洛芬克隆菌株转座子插入突变体的构建[J].西北农林科技大学学报(自然科学版),2016,44(4):135-141.
[9]吴程华,严玉霖,高洪,等.Red同源重组技术在大肠埃希菌基因敲除中的应用[J].上海畜牧兽医通讯,2015,1:9-11.
[10]曾吉祥.Red/ET同源重组技术在载体构建中的应用[J].生物技术世界,2015,7:232-233.
[11]刘陆罡,纪晓俊,沈梦秋,等.大肠杆菌无痕重组的策略与应用[J].中国生物工程杂志,2014,34(8):88-96.
[12]Murphy K C.Use of bacteriophage lambda recombination functions to promote gene replacement in Escherichia coli[J].Bact Eriol,1998,180(8):2063-2071.
[13]Yu D,Ellis H M,Lee E,et al.An efficient recombination system for chromosome engineering in Escherichia coli[J].Proceedings of the National Academy of Sciences,2000,97(97):5978-5983.
[14]Datsenko K A,Wanner B L.One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products[J].Proceedings of the National Academy of Sciences of the United States of America,2000,97(12):6640-6645.
[15]Lee D J,Bingle L E,Heurlier K,et al.Gene doctoring:A method for recombineering in laboratory and pathogenic Escherichia coli strains[J].BMC Micro biology,2009,9(1):131.
[16]Jeong J,Cho N,Jung D,et al.Genome-scale genetic engineering in Escherichia coli[J].Biotechnology Advances,2013,31(6):804-810.
[17]Nafissi N,Slavcev R.Bacteriophage recombination systems and biotechnical applications[J].Applied Microbiology and Biotechnology,2014,98(7):2841-2851.
[18]Egan M.Lambda Red-mediated recombineering in the attaching and effacing pathogen Escherichia albertii[J].Biological Procedures Online,2016,18(1):1-13.
[19]Poteete A R.What makes the bacteriophageλRed system useful for genetic engineering:Molecular mechanism and biological function[J].FEMS Microbiology Letters,2001,201(1):9-14.
[20]Karakousis G,Ye N,Li Z,et al.The beta protein of phage lambda binds preferentially to an intermediate in DNA renaturation[J].Journal of Molecular Biology,1998,276(4):721-731.
[21]Li Z,Karakousis G,Chiu S K,et al.The beta protein of phage lambda promotes strand exchange[J].Journal of Molecular Biology,1998,276(4):733-744.
[22]Kovall R,Matthews B W.Toroidal structure of lambda-exonuclease[J].Science,1997,277(5333):1824-1827.
[23]王锴,张志强,朱春红,等.致鹅卵黄性腹膜炎大肠杆菌cya、crp双基因缺失株构建及部分生物学特性分析[J].中国兽医学报,2013,33(9):49-54.
[24]Krishnan S T,Moolman M C,Laar T V,et al.Essential validation methods for E.coli strains created by chromosome engineering[J].Journal of Biological Engineering,2015,9(1):1-14.
[25]Passy S I,Yu X,Li Z,et al.Rings and filaments of beta protein from bacteriophage lambda suggest a superfamily of recombination proteins[J].Proceedings of the National Academy of Sciences of the United States of America,1999,96(8):4279-4284.
[26]Poteete A R,Fenton A C,Murphy K C.Roles of RuvC and RecG in PhageλRed-mediated recombination[J].Journal of Bacteriology,1999,181(17):5402-5408.
[27]原志伟.F18大肠杆菌黏附素菌体表面功能性展呈及其染色体-质粒平衡致死系统的构建[D].扬州:扬州大学,2007.
[28]吕沈聪,赵颖颖,钟卫鸿.Red同源重组在大肠杆菌基因敲除中的应用[J].化学与生物工程,2013,30(6):1-6.
[29]程毅鹏,饶志明,杨套伟,等.一种新型抗性质粒的构建及其在核黄素生产菌中的运用[J].应用与环境生物学报,2015,21(3):435-440.
[30]王少辉.禽致病性大肠杆菌DE205B黏附及侵袭相关因子的致病作用[D].南京:南京农业大学,2011.
[31]胡逢雪,丁锐,崔震海,等.大肠杆菌基因无痕敲除技术及策略[J].生物技术通讯,2013,4:552-557.
[32]Tischer B K,Von E J,Kaufer B,et al.Two-step redmediated recombination for versatile high-efficiency markerless DNA manipulation in Escherichia coli[J].Biotechniques,2006,40(40):191-197.
[33]Lalioti M D,Heath J K.A new method for generating point mutations in bacterial artificial chromosomes by homologous recombination in Escherichia coli[J].Nucleic Acids Research,2001,29(3):E14.
[34]Madyagol M,Al-Alami H,Levarski Z,et al.Gene replacement techniques for Escherichia coli genome modification[J].Folia Microbiologica,2011,56(3):253-263.
[35]Kolisnychenko V,Plunkett G,Herring C D,et al.Engineering a reduced Escherichia coli genome[J].Genome Research,2002,12(4):640-647.
[36]Herring C D,Glasner J D,Blattner F R.Gene replacement without selection:Regulated suppression of amber mutations in Escherichia coli[J].Gene,2003,311(2):153-163.
[37]Yang J,Sun B,Huang H,et al.Multiple-site genetic modifications in Escherichia coli using lambda-Red recombination and I-Sce I cleavage[J].Biotechnology Letters,2015,37(10):1-8.
[38]王瑶,许杨,陈楠,等.在大肠杆菌中利用SCLM系统进行高效率λ-Red基因敲除/整合的新策略[J].微生物学通报,2015,42(4):699-711.
[39]葛高顺,张立超,赵昕,等.大肠杆菌基因组基因无痕敲除的优化方法[J].中国生物工程杂志,2014,34(6):68-74.
[40]Reisch C R,Prather K L J.The no-SCAR(Scarless Cas9 Assisted Recombineering)system for genome editing in Escherichia coli[J].Scientific Reports,2015,5:15096.
[41]Mizoguchi H,Tanakamasuda K,Mori H.A simple method for multiple modification of the Escherichia coli K-12chromosome[J].Bioscience,Biotechnology,and Biochemistry,2007,71(12):2905-2911.
[42]潘虹.产志贺毒素大肠杆菌毒素基因敲除及毒力测定的研究[D].兰州:甘肃农业大学,2012.
[43]陈燕杰.marA基因对鸭大肠杆菌多重耐药的调控作用[D].郑州:河南农业大学,2012.
[44]Liang R,Liu J.Scarless and sequential gene modification in Pseudomonas using PCR product flanked by short homology regions[J].BMC Microbiology,2010,10(1):209.
[45]吕雪莲,于申业,倪宏波,等.通过改进Red重组方法快速构建肠炎沙门菌毒力岛SPI-1缺失株[J].中国预防兽医学报,2014,36(11):852-855.
[2]Wang S.DotU expression is highly induced during in vivoinfection and responsible for virulence and Hcp1secretion in avian pathogenic Escherichia coli[J].Frontiers in Microbiology,2014,5:588.
[3]Wang S.IbeR facilitates stress-resistance,invasion and pathogenicity of avian pathogenic Escherichia coli[J].PLoS One,2015,10(3):e0119698.
[4]李继东,才学鹏.山羊痘病毒TK基因功能缺失对其增殖的影响[J].中国畜牧兽医,2017,44(2):319-326.
[5]赵雪丽,闫若潜,吴志明,等.鉴别猪伪狂犬病病毒gE基因缺失疫苗和野毒感染的二重PCR诊断方法的建立和应用[J].中国畜牧兽医,2015,42(4):865-870.
[6]陈柳,余斌,倪征,等.表达小鹅瘟病毒VP2蛋白重组鸭瘟病毒的构建及其生物学特性[J].中国农业科学,2016,49(14):2813-2821.
[7]周化民,孙旭利,陈建明.同源重组和重组酶Rad51的调控[J].生物化学与生物物理进展,2016,12:1154-1162.
[8]卫亚红,段敏,向照举,等.降解布洛芬克隆菌株转座子插入突变体的构建[J].西北农林科技大学学报(自然科学版),2016,44(4):135-141.
[9]吴程华,严玉霖,高洪,等.Red同源重组技术在大肠埃希菌基因敲除中的应用[J].上海畜牧兽医通讯,2015,1:9-11.
[10]曾吉祥.Red/ET同源重组技术在载体构建中的应用[J].生物技术世界,2015,7:232-233.
[11]刘陆罡,纪晓俊,沈梦秋,等.大肠杆菌无痕重组的策略与应用[J].中国生物工程杂志,2014,34(8):88-96.
[12]Murphy K C.Use of bacteriophage lambda recombination functions to promote gene replacement in Escherichia coli[J].Bact Eriol,1998,180(8):2063-2071.
[13]Yu D,Ellis H M,Lee E,et al.An efficient recombination system for chromosome engineering in Escherichia coli[J].Proceedings of the National Academy of Sciences,2000,97(97):5978-5983.
[14]Datsenko K A,Wanner B L.One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products[J].Proceedings of the National Academy of Sciences of the United States of America,2000,97(12):6640-6645.
[15]Lee D J,Bingle L E,Heurlier K,et al.Gene doctoring:A method for recombineering in laboratory and pathogenic Escherichia coli strains[J].BMC Micro biology,2009,9(1):131.
[16]Jeong J,Cho N,Jung D,et al.Genome-scale genetic engineering in Escherichia coli[J].Biotechnology Advances,2013,31(6):804-810.
[17]Nafissi N,Slavcev R.Bacteriophage recombination systems and biotechnical applications[J].Applied Microbiology and Biotechnology,2014,98(7):2841-2851.
[18]Egan M.Lambda Red-mediated recombineering in the attaching and effacing pathogen Escherichia albertii[J].Biological Procedures Online,2016,18(1):1-13.
[19]Poteete A R.What makes the bacteriophageλRed system useful for genetic engineering:Molecular mechanism and biological function[J].FEMS Microbiology Letters,2001,201(1):9-14.
[20]Karakousis G,Ye N,Li Z,et al.The beta protein of phage lambda binds preferentially to an intermediate in DNA renaturation[J].Journal of Molecular Biology,1998,276(4):721-731.
[21]Li Z,Karakousis G,Chiu S K,et al.The beta protein of phage lambda promotes strand exchange[J].Journal of Molecular Biology,1998,276(4):733-744.
[22]Kovall R,Matthews B W.Toroidal structure of lambda-exonuclease[J].Science,1997,277(5333):1824-1827.
[23]王锴,张志强,朱春红,等.致鹅卵黄性腹膜炎大肠杆菌cya、crp双基因缺失株构建及部分生物学特性分析[J].中国兽医学报,2013,33(9):49-54.
[24]Krishnan S T,Moolman M C,Laar T V,et al.Essential validation methods for E.coli strains created by chromosome engineering[J].Journal of Biological Engineering,2015,9(1):1-14.
[25]Passy S I,Yu X,Li Z,et al.Rings and filaments of beta protein from bacteriophage lambda suggest a superfamily of recombination proteins[J].Proceedings of the National Academy of Sciences of the United States of America,1999,96(8):4279-4284.
[26]Poteete A R,Fenton A C,Murphy K C.Roles of RuvC and RecG in PhageλRed-mediated recombination[J].Journal of Bacteriology,1999,181(17):5402-5408.
[27]原志伟.F18大肠杆菌黏附素菌体表面功能性展呈及其染色体-质粒平衡致死系统的构建[D].扬州:扬州大学,2007.
[28]吕沈聪,赵颖颖,钟卫鸿.Red同源重组在大肠杆菌基因敲除中的应用[J].化学与生物工程,2013,30(6):1-6.
[29]程毅鹏,饶志明,杨套伟,等.一种新型抗性质粒的构建及其在核黄素生产菌中的运用[J].应用与环境生物学报,2015,21(3):435-440.
[30]王少辉.禽致病性大肠杆菌DE205B黏附及侵袭相关因子的致病作用[D].南京:南京农业大学,2011.
[31]胡逢雪,丁锐,崔震海,等.大肠杆菌基因无痕敲除技术及策略[J].生物技术通讯,2013,4:552-557.
[32]Tischer B K,Von E J,Kaufer B,et al.Two-step redmediated recombination for versatile high-efficiency markerless DNA manipulation in Escherichia coli[J].Biotechniques,2006,40(40):191-197.
[33]Lalioti M D,Heath J K.A new method for generating point mutations in bacterial artificial chromosomes by homologous recombination in Escherichia coli[J].Nucleic Acids Research,2001,29(3):E14.
[34]Madyagol M,Al-Alami H,Levarski Z,et al.Gene replacement techniques for Escherichia coli genome modification[J].Folia Microbiologica,2011,56(3):253-263.
[35]Kolisnychenko V,Plunkett G,Herring C D,et al.Engineering a reduced Escherichia coli genome[J].Genome Research,2002,12(4):640-647.
[36]Herring C D,Glasner J D,Blattner F R.Gene replacement without selection:Regulated suppression of amber mutations in Escherichia coli[J].Gene,2003,311(2):153-163.
[37]Yang J,Sun B,Huang H,et al.Multiple-site genetic modifications in Escherichia coli using lambda-Red recombination and I-Sce I cleavage[J].Biotechnology Letters,2015,37(10):1-8.
[38]王瑶,许杨,陈楠,等.在大肠杆菌中利用SCLM系统进行高效率λ-Red基因敲除/整合的新策略[J].微生物学通报,2015,42(4):699-711.
[39]葛高顺,张立超,赵昕,等.大肠杆菌基因组基因无痕敲除的优化方法[J].中国生物工程杂志,2014,34(6):68-74.
[40]Reisch C R,Prather K L J.The no-SCAR(Scarless Cas9 Assisted Recombineering)system for genome editing in Escherichia coli[J].Scientific Reports,2015,5:15096.
[41]Mizoguchi H,Tanakamasuda K,Mori H.A simple method for multiple modification of the Escherichia coli K-12chromosome[J].Bioscience,Biotechnology,and Biochemistry,2007,71(12):2905-2911.
[42]潘虹.产志贺毒素大肠杆菌毒素基因敲除及毒力测定的研究[D].兰州:甘肃农业大学,2012.
[43]陈燕杰.marA基因对鸭大肠杆菌多重耐药的调控作用[D].郑州:河南农业大学,2012.
[44]Liang R,Liu J.Scarless and sequential gene modification in Pseudomonas using PCR product flanked by short homology regions[J].BMC Microbiology,2010,10(1):209.
[45]吕雪莲,于申业,倪宏波,等.通过改进Red重组方法快速构建肠炎沙门菌毒力岛SPI-1缺失株[J].中国预防兽医学报,2014,36(11):852-855.