文献类型：期刊文献

中文题名：红色糖多孢菌红霉素抗性基因的克隆与鉴定

英文题名：Cloning and Identification of Antibiotic Resistance Gene of Saccharopolyspora Erythraea

作者：孙丽萍[1];沈琼[1];叶江[1];吴海珍[1];张惠展[1]

机构：[1]华东理工大学生物反应器工程国家重点实验室,上海200237

年份：2002

卷号：28

期号：1

起止页码：20

中文期刊名：华东理工大学学报（自然科学版）

外文期刊名：Journal of East China University of Science and Technology

收录：CSTPCD;;EI(收录号：2002397116412);Scopus;北大核心:【北大核心2000】；CSCD:【CSCD2011_2012】；

语种：中文

中文关键词：鸟枪法;原生质体转化;红色糖多孢菌;红霉素抗性基因;双重抗性筛选;基因克隆;基因鉴定

外文关键词：shot gun; protoplast transformation; Saccharopolyspora erythraea; erythromycin resistance gene; double resistance screen

摘要：将红色糖多孢菌 ( Saccharopolyspora erythraea)的染色体 DNA用 Pst I酶切后与载体p UWL2 0 1连接 ,转化变铅青链霉菌 ( S.lividans) TK2 3的原生质体。采用双重抗性筛选得到了 9个转化子。分别抽提其中的质粒并再次转化原生质体 ,在含红霉素的平板上各筛选约 2 0 0个转化子 ,其中有 3个在抗性板上生长良好 ,分别命名为 p LP42、p LP1 b和 p LP44。对其中的 p LP42酶切分析表明 ,其含有约 3.0 kb的红色糖多孢菌 ( Saccharopolyspora erythraea)染色体 DNA片段 ,它的载体部分发生了缺失。以 p UC1 8为载体 ,将 p LP42的 1 .7kb- Kpn I片断克隆、测序、经与 Genebank的erm E基因顺序比较 ,证实已克隆了 Saccharopolyspora
Saccharopolyspora erythraea chromosomal DNA digested extensively with PstI has been cloned in shuttle vector pUWL201, which is transformed into protoplast of Streptomyces lividans TK23. Nine transformants are obtained by double antibiotic resistant screening. The nine plasmids are extracted and re transformed, then about 200 transformants are screened on plate containing erythromycin, respectively. It is found that three transformants grow well on resistant plate, designated pLP42, pLP1b and pLP44, respectively. With the electrophresis analysis of restriction fragments of pLP42, our experiment shows that pLP42 contains about 3.0kb foreign fragment from S.erythraea chromosomal DNA, and a section of vector pUWL201 has been deleted. A 1.7kb KpnI fragment from pLP42 has been cloned and sequenced. Compared with the ermE sequence in genebank, the cloned S.erythraea chromosome fragment firmly contains the antibiotic resistance gene.