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Red/ET重组AHBA生物合成基因簇打靶载体的构建与鉴定 被引量：1

Construction and Identification of 3-Amino-5-hydroxybenzoic Acid(AHBA) Biosynthesis Gene Cluster Targeting Vector in Streptomyces by Red/ET Recombination
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摘要 为确定3-氨基-5-羟基-苯甲酸(AHBA)生物合成基因簇在链霉菌中与次生代谢产物的关系,运用PCR技术,从33株AHBA合酶基因阳性菌株扩增与AHBA生物合成基因簇中编码AHBA合酶(A)、氧化还原酶(O)、磷酸化酶(P)基因,获得24株AOP基因阳性菌株.根据靶基因A基因下游和P基因上游同源序列设计50 bp引物,中间插入卡那霉素抗性基因的DNA片段,进行PCR,获得外源DNA片段.经过电转化,将外源DNA片段和pKC1139-AOP重组质粒共转入含重组酶质粒大肠杆菌HS996/pSC101-BAD-gba-(Tet).在Red重组酶的作用下,外源DNA片段与重组质粒pKC1139-AOP上的AHBA基因簇的同源区域重组,构建了AHBA基因簇打靶载体.研究显示了Red/ET重组工作效率高、操作简单、精确的优点,可大大缩短构建打靶载体的时间. In order to identify the secondary metabolite related to AHBA biosynthetic gene cluster in streptomyces strains 24 positive strains harboring the linked genes encoding AHBA synthase（A）,oxidoreductase（O） and phosphatase（P） were acquired from 33 AHBA synthase positive streptomyces strains.Kanamycin-resistant gene flanked by homologous fragments of AHBA synthase and phosphatase genes（50 bp） was amplified by PCR and co-electrotransformed with the recombinant plasmid pKC1139-AOP into E.coli HS996/pSC101-BAD-gba-（Tet）. A Red/lET recombinant plasmid with kanamycin-resistant gene, pKC1139-AOP-Km, for targeting the AHBA biosynthesis gene cluster was constructed. The results showed that Red/ET recombination has the advantage of high efficiency and time saving for construction of the targeting vector in studies of the gene function in streptomyces.
作者 王广林 张会图 张金池 王以光
机构地区 南京林业大学森林资源与环境学院 皖西学院化学与生命科学系 中国协和医科大学医药生物技术研究所
出处 《安徽师范大学学报（自然科学版）》 CAS 北大核心 2009年第6期569-574,共6页 Journal of Anhui Normal University(Natural Science)
基金 国家自然科学基金项目(30570039) 国家"十五"科技攻关项目
关键词 Red/ET重组 AHBA基因簇 打靶载体 链霉菌 Red/ET recombination AHBA gene cluster targeting vector streptomyces
分类号 Q78 [生物学—分子生物学]
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参考文献13

  • 1ZHANG Y, BUCHHOLZ F, MUYRERS J P P, et al. A new logic for DNA engineering using recom-bination in Escherichia coli[J]. Nat C, enet, 1998,20(2) : 123 - 128.
  • 2JOEP P P. MUYRERS Youming Zhang, FRAENK Buchholz, A. Francis Stewart. RecE/RecT and Red/Red initiate double-stranded break repair by specifically interacting with their respective partners[J]. Genes Dev, 2000,14:1971 - 1982.
  • 3KATZ L, DONADIO S. Polyketide synthesis: prospects for hybrid antibiotics[J]. Annu Rev Microbiol, 1993,47 : 875 - 912.
  • 4BECKER A M, HERLT A J, HILTON G L, et al. 3-Amino-5-hydroxybenzoic acid in antibiotic biosynthesis. Ⅵ. Directed biosynthesis studies with ansamycin antibiotics[J]. J Antibiot, 1983,36:1323 - 1328.
  • 5GHISALBA O, NUESCH J. A genetic approach to the biosynthesis of the rifamycin-chromophore in Nocardia mediterranei. Ⅳ. Identification of 3-arnino-5-hydroxybenzoic acid as a direct precursor of the seven-carbon amino starter-unit[J]. J Antibiot, 1981,34:64 - 71.
  • 6YU T W, MULLER R, MULLER M, et al. Mutational analysis and reconstituted expression of the biosynthetic genes involved in the formation of 3-amino-5-hydroxybenzoic acid, the starter unit of rifamycin biosynthesis in amycolatopsis Mediterranei S699 [J]. J Biol Chem, 2001,276(16) : 12546 - 12555.
  • 7ZHANG HT, WU LZ, WANG YG, et al. PCR screening of 3-amino-5-hydroxybenzonic acid synthase gene leads to identification of ansamycins and AHBA-related antibiotic producers in Actinomycetes[J]. J Appl Microbio, 2009,1364- 1372.
  • 8ZHANG YM, MUYRERS J P P, RIENTJES J, STEWART A F. Phage annealing proteins promote oligonucleotide-directed mutagenesis in Escherichia coli and mouse ES cells[J]. BMC Mol Biol, 2003,16( 1 ) : 1 - 14.
  • 9BIERMAN M, LOGAN R, O' BRIEN K, et al. Plasmid cloning vectors for the conjugal transfer of DNA from Escherichia coil to Streptomyces spp[J]. Gene, 1992,116:43 - 49.
  • 10KIRILL A D, BARRY L W. One-step inactivation of chromosomal genes in Escherichia coliK - 12 using PCR products[J]. Proc Natl Acad Sci USA, 2000.97 (12) : 6640 - 6645.

同被引文献18

  • 1赫卫清,李京艳,孙桂芝,刘玉瑛,王以光.反相高效液相谱型分析检测吸水链霉菌17997变株发酵新产物[J].中国抗生素杂志,2006,31(3):168-171. 被引量：5
  • 2Davies J,Yagisawa M.Biochemistry and genetic regulation of commercially important antibiotics[M].In:Vining L C(ed),Addison-Wesley,London,1983,329-354.
  • 3Katz L,Donadio S.Polyketide synthesis:prospects for hybrid antibiotics[J].Annual Review of Microbiology,1993,47:875-912.
  • 4Becker A M,Herlt A J,Hilton G L,et al.3-Amino-5-hydroxybenzoic acid in antibiotic biosynthesis VI.directed biosynthesis studies with ansamycin antibiotics[J].J Antibiot,1983,36:1323-1328.
  • 5Yu T W,Muller R,Muller M,et al.Mutational analysis and reconstituted expression of the biosynthetic genes involved in the formation of 3-amino-5-hydroxybenzoic acid,the starter unit of rifamycin biosynthesis in Amycolatopsis mediterranei S699[J].J Biol Chem,2001,276(16):12546-12555.
  • 6August P R,Tang L,Yoon Y J,et al.Biosynthesis of the ansamycin antibiotic rifamycin:deductions from the molecular analysis of the rif biosynthetic gene cluster of Amycolatopsis mediterranei S699[J].Chemistry & Biology,1998,5:69-79.
  • 7Chen S,Von B D,Hale V,et al.Biosynthesis of ansatrienin (mycotrienin) and naphthomycin.Identification and analysis of two separate biosynthetic gene clusters in Streptomyces collinus T 1892[J].Eur J Biochem,1999,261:98-107.
  • 8Kim C G,Lamichhane J,Song K I,et al.Biosynthesis of rubradirin as an ansamycin antibiotic from Streptomyces achromogenes var.rubradiris NRRL3061[J].Arch Microbiol,2008,189:463-473.
  • 9He W Q,Wu L Z,Gao Q J,et al.Identification of AHBA biosynthetic genes related to geldanamycin biosythesis in Streptomyces hygroscopicus 17997[J].Curr Microbiol,2006,52:197-203.
  • 10Yu T W,Bai L,Clade D,et al.The biosynthetic gene cluster of the maytansinoid antitumor agent ansamitocin from Actinosynnema pretiosum[J].Proc Natl Acad Sci USA,2002,99:7968-7973.

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安徽师范大学学报（自然科学版）
2009年 第6期

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