淀粉酶链霉菌BAC基因组文库的构建及鉴定  

Construction and Identification of BAC Genomic Library from Streptomyces diastaticus

作　　者：侯松涛[1] 王波[1] 张贝贝[1] 葛亚东[1,2] 王宝娟[1,2] 朱国萍[1,2] 

机构地区：[1]安徽师范大学生命科学学院分子进化重点实验室,安徽芜湖241000 [2]安徽师范大学分子生物学及生物技术研究所,安徽芜湖241000

出　　处：《激光生物学报》2009年第2期161-165,共5页Acta Laser Biology Sinica

基　　金：国家自然科学基金(30500300);国家教育部“新世纪优秀人才支持计划”(NCEFT-06-0558);安徽省优秀青年科技基金(06043089);安徽省引进海外留学人才基金(NO.2005Z032);安徽省教育厅自然科学基金重点项目(2006KJ061A);重要生物资源保护与利用研究安徽省重点实验资助

摘　　要：提取淀粉酶链霉菌SM33基因组DNA，用Hind III部分酶解后回收40kh～60kb大小的高分子量DNA，与质粒载体pIndigoBAC536连接，电击转化EP1300感受态细胞，经蓝自斑筛选共挑取了5184个白色克隆。从文库中随机挑选10个克隆，酶切检测平均插入片段约为50kh，覆盖了32．4倍基因组。并且插入片段均具有9～15个Not I酶切位点，符合链霉菌基因组特征。通过对BAC文库的筛选，获得苹果酸脱氢酶基因（mdh）的保守序列，与已知的链霉菌mdh具有很高的相似性。淀粉酶链霉菌BAC基因组文库的建立，对基因克隆、基因组物理图谱、次级代谢途径、新抗生素的发现以及工业用酶的应用等研究均有重要意义。The genomic DNA of Streptomyces diastaticus SM33 was extracted and partially digested by Hind III. The 40 - 60 kb high molecular weight DNA was extracted and ligated to pIndigoBAC536. The ligation mixture was transformed into EPI300 competent cells. A total of 5 184 colonies were obtained by white-blue screening. The analysis of enzyme digestion showed that the average size of the insert fragments is about 50 kb, covering 32.4 x haploid genome equivalents. And 9 - 15 Not I restriction site in the insert fragments, which is consistent with the features of Streptomyces gehome. The conserved sequence of mdh gene encoding malate dehydrogenase was acquired by BAC library screening, which exhibited very high similarity with known mdh genes from Streptomyces. These results suggest that this BAC library provide useful tools for the researches on gene cloning, genome physical mapping, the secondary metabolic pathways, new antibiotics and industrial enzymes.

关 键 词：淀粉酶链霉菌 基因组 细菌人工染色体(BAC)文库 

分 类 号：Q78[生物学—分子生物学]