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重组工程法敲除恶臭假单胞菌KT2440的染色体基因 被引量：2

Recombineering Mediated Chromosal Gene Knockout of Pseudomonas Putida KT2440
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摘要 恶臭假单胞菌KT2440是在生物降解环境有机物和表达异源基因等方面具重要作用的环境微生物.基因敲除是研究基因和蛋白功能的必要手段.常规的基因敲除方法往往会烦琐耗时且易引入突变的基因克隆操作.本研究采用重组工程(即重组酶催化的PCR产物与基因组上同源片段之间的同源重组)来实现恶臭假单胞菌KT2440的基因敲除.本方法高效、简便、快捷且可以同时进行多个基因的操作,可成为通用的恶臭假单胞菌KT2440的遗传研究方法. Pseudomonas putida KT2440 plays an important role in the degradation of environmental organic compounds and it is a good host for foreign gene expression.Gene knockout is essential to the investigations of genes and proteins.Classic chromosal gene knockout methods usually involve the gene cloning steps,which are often tedious,time consuming and tend to introduce mutations.In this paper,a recombineering strategy characterized by the homologous recombination between the PCR product and the genome,is used for the P.putida KT2440 gene knockout.This strategy is highly efficient,convenient,rapid and amendable for the simultaneously manipulation of many genes,and can be used as a general gene knockout method.
作者 杨运文 蒋伏欢 宋杰 尚广东
机构地区 南京师范大学生命科学学院江苏省功能微生物与功能基因组学重点实验室
出处 《南京师大学报（自然科学版）》 CAS CSCD 北大核心 2011年第4期96-101,共6页 Journal of Nanjing Normal University(Natural Science Edition)
基金 "重大新药创制"科技重大专项课题(2009ZX091032674)
关键词 恶臭假单胞菌KT2440 基因敲除 重组工程 Pseudomonas putida KT2440 gene knockout recombineering
分类号 Q819 [生物学—生物工程]
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参考文献12

  • 1Timmis K. Pseudomonas putida : a cosmopolitan opportunist par excellence [ J ]. Environ Microbial, 2002,4 ( 12 ) :779-781.
  • 2Jimenez J, Jose B, Garcia L, et al. Genomic analysis of the aromatic catabolic pathways from Pseudomonas putida KT2440 [ J ]. Environ Mierobio1,2002,4 (12) : 824-841.
  • 3Nelson K, Weinel C, Paulsen I, et al. Complete genome sequence and comparative analysis of the metabolically versatile Pseudomonas putida KT2440 [ J ]. Environ Mierobiol, 2002,4 ( 12 ) : 799 -808.
  • 4Lee Y, Oh S, Park W. Inactivation of the Pseudomonas putida KT2440 dsbA gene promotes extracellular matrix production and biofilm formation[ J]. FEMS Mierobiol Lett ,2009,297 ( 1 ) :38-48.
  • 5Kim J, Yeom J, Jean C, et al. Intracellular 2 - keto - 3 - deoxy - 6 - phosphogluconate is the signal for carbon eatabolite re- pression of phenylacetic acid metabolism in Pseudomonas putida KT2440 [ J ]. Microbiology ,2009,155 (7) :2 420-2 428.
  • 6Zhang Y, Buchholz F P, Stewart A. A new logic for DNA engineering using recombination in Escherichia coli[ J ]. Nat Gen- et, 1998,20 (2) : 123-128.
  • 7Sharan S K, Thomason L C, Kuznetsov S G et al. Recombineering: a homologous recombination-based method of genetic en- gineering [ J ]. Nat Protoc, 2009,4 ( 2 ) : 206-223.
  • 8Karlinsey J E, Hughes K T. Genetic transplantation: Salmonella enterica serovar Typhimurium as a host to study sigma factor and anti-sigma factor interactions in genetically intractable systems[ J]. J Bacterio1,2006,188 (1) :103-114.
  • 9van Kessel J C, Haffull G F. Efficient point mutagenesis in mycobacteria using single-stranded DNA recombineering : charac- terization of antimycobacterial drug targets [ J ]. M ol Microbio1,2008,67 ( 5 ) : 1 094-1 107.
  • 10Sambrook J, Fritsch E F, Maniatis T. Molecular cloning: a laboratory manual[ M]. 2nd ed. New York: Cold Spring Har- bor Laboratory Press, 1989:16-34.

同被引文献21

  • 1WARMING S, COSTANTINO N, COURT D L, et al. Simple and highly efficient BAC recombineering using galK selec, tion [J]. Nucleic Acids Res,2005,33(4) :e36.
  • 2WONG Q N, NG V C, LIN M C, et al. Efficient and seamless DNA recombineering using a thymidylate synthase a selectionsystem in Escherichia coli[J]. Nucleic Acids Res, 2005,33(6) : e59.
  • 3LI X T, THOMASON L C, SAWITZKE J A, et al. Positive and negative selection using the tetA-sacB cassette : recombineer- ing and P1 transduction in Escherichia coli[J]. Nucleic Acids Res, 2013,41 (22) : e204.
  • 4GREGG C J, LAJOIE M J, NAPOLITANO M G, et al. Rational optimization of tolC as a powerful dual selectable marker for genome engineering[J]. Nucleic Acids Res,2014,42(7) :4 779-4 790.
  • 5HARTLEY J L, TEMPLE G F, BRASCH M A. DNA cloning using in vitro site-specific recombination [J ]. Genome Res, 2000, 10(11):1 788-1 795.
  • 6HOUSE B L, MORTIMER M W, KAHN M L. New recombination methods for Sinorhizobium meliloti genetics[J]. Appl Envi- ron Microbiol, 2004,70(5 ) : 2 806-2 815.
  • 7WANG H, BIAN X, XIA L, et al. Improved seamless mutagenesis by recombineering using ccdB for counterselection [J]. Nu- cleic Acids Res, 2014,42 (5) : e37.
  • 8BERNARD P, COUTURIER M. Cell killing by the F plasmid CcdB protein involves poisoning of DNA-topoisomerase II com- plexes [ J ]. J Mol Biol, 1992,226 (3) : 735-745.
  • 9DATSENKO K A, WANNER B L. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products [ J ]. Proc Natl Acad Sei USA, 2000,97 (12) :6 640-6 645.
  • 10LI M Z, ELLEDGE S J. Magic, an in vivo genetic method for the rapid construction of recombinant DNA molecules [J]. Nat Genet,2005,37(3) :311-319.

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南京师大学报（自然科学版）
2011年 第4期

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