| Red/ET重组AHBA生物合成基因簇打靶载体的构建与鉴定 |
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| 引用本文： | 王广林,张会图,张金池,王以光.Red/ET重组AHBA生物合成基因簇打靶载体的构建与鉴定[J].安徽师范大学学报(自然科学版),2009,32(6):569-574. |
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| 作者姓名： | 王广林 张会图 张金池 王以光 |
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| 作者单位： | 南京林业大学,森林资源与环境学院,江苏,南京,210039;皖西学院,化学与生命科学系,安徽,六安,237012;中国协和医科大学,医药生物技术研究所,北京,100050;南京林业大学,森林资源与环境学院,江苏,南京,210039 |
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| 基金项目： | 国家自然科学基金项目，国家"十五"科技攻关项目 |
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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.
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| 关 键 词： | Red/ET重组 AHBA基因簇 打靶载体 链霉菌 |
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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| WANG Guang-lin,ZHANG Hui-tu,ZHANG Jin-chi,WANG Yi-guang.Construction and Identification of 3-Amino-5-hydroxybenzoic Acid(AHBA) Biosynthesis Gene Cluster Targeting Vector in Streptomyces by Red/ET Recombination[J].Journal of Anhui Normal University(Natural Science Edition),2009,32(6):569-574. |
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| Authors: | WANG Guang-lin ZHANG Hui-tu ZHANG Jin-chi WANG Yi-guang |
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| Institution: | WANG Guang-lin1,3,ZHANG Hui-tu2,ZHANG Jin-chi1,WANG Yi-guang2(1.College of Forest Resouces , Environment,Nanjing Forestry University,Nanjing 210037,China,2.Institute of Medicinal Biotechnology,Peking Union Medical Colleg,Beijing 100050,3.Department of Chemistry , Life Science,West Anhui University,Lu\'an 237012,China) |
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| Abstract: | 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-... |
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| Keywords: | Red/ET recombination AHBA gene cluster targeting vector streptomyces |
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