文献类型：期刊文献

中文题名：林可链霉菌中的同源重组

英文题名：HOMOLOGOUS RECOMBINATION IN STERPTOMYCES LINCOLNENSIS B48

作者：唐雅珺[1];吴海珍[1];叶江[1];张惠展[1]

机构：[1]华东理工大学国家生物反应器工程国家重点实验室,上海200237

年份：2001

卷号：41

期号：5

起止页码：559

中文期刊名：微生物学报

外文期刊名：Acta Microbiologica Sinica

收录：CSTPCD;;Scopus;北大核心:【北大核心2000】；CSCD:【CSCD2011_2012】；PubMed;

基金：国家自然科学基金资助项目 ( 2 94 762 35)

语种：中文

中文关键词：林可链霉菌;同源重组;同源整合;同源交换;抗生素生产菌

外文关键词：Streptomyces lincolnensis, Homologous recombination, Homologous integration, Homo^logous exchange

摘要：为研究链霉菌中的同源整合频率和机制 ,采用不能在链霉菌中复制的大肠杆菌质粒转化链霉菌StreptomyceslincolnensisB48。质粒pYYE0 4a1上携带的被硫链丝菌素抗性基因灭活的林可霉素生物合成基因与染色体DNA上的同源基因发生重组 ,经过低抗筛选 ,得到两个突变子S .lincolnensisYY1和S .lincolnensisYY2。进一步以硫链丝菌素抗性基因为探针杂交染色体DNASmaⅠ片段 ,S .lincolnensisYY1和S .lincolnensisYY2都得到 1 5kb的阳性条带 ;而以缺失的lacZ基因为探针杂交染色体DNAHindⅢ和SmaⅠ联合酶切片段 ,只有S .lincolnensisYY2得到 4 4kb的阳性条带。Southern杂交结果表明S .lincolnensisYY1是由同源交换或二次重组产生的 ,而S .lincolnensisYY2为同源整合的结果。为验证同源整合子上大肠杆菌复制子和氨苄抗性基因的存在 ,用SphⅠ酶切染色体DNA后连接 ,连接液转化E .coliJM83感受态细胞 ,在氨苄抗性板上得到 2个转化子 ,命名为pSLE1。对其进行酶切鉴定的结果表明它是转化质粒pYYE0
To study frequency and mechanism of homologous recombination in Streptomyces, an E.coli plasmid which cannot replicate in Streptomyces was transformed into Streptomyces lincolnensis B48.After homologous recombination between delta lincomycin biosynthetic genes inactivated by thiostrepton resistant gene ( ts\+r ) carried on pYYE04a1 and homologous sequences on the chromosome, S.lincolnensis YY1 and S.lincolnensis YY2 were obtained on SMA with low thiostrepton concentration.Hybridization of chromosomal DNA samples of S.lincolnensis YY1, S.lincolnensis YY2,standard S.lincolnensis and S.lincolnensis YYc digested with Sma I with the probe of ts\+r gene gave signal corresponding to a fragment of 1.5kb in the former two;Nevertheless,hybridization of chromosomal DNA digested with Hin dⅢ and SmaⅠ using the probe of Δlac Z′ gene resulted in positive fragment of 4.4kb only in S.lincolnensis YY2.Southern hybridizations indicate that S.lincolnensis YY1 is the result of homologous exchange while S.lincolnensis YY2 comes from bomologous recombination.To prove the existence of E.coli replicon and ampicillin resistant gene on the chromosome of S.lincolnensis YY2,its DNA digested with Sph I was ligated and then transformed into E.coli JM83 competent cell.Two transformants named pSLE1 grew on the plate containing ampicillin.It's confirmed that pSLE1 is a part of pYYE04a1 from its digestion with different enzymes.