【摘要】 提取淀粉酶链霉菌SM33基因组DNA,用HindIII部分酶解后回收40kb～60kb大小的高分子量DNA,与质粒载体pIndigoBAC536连接,电击转化EPI300感受态细胞,经蓝白斑筛选共挑取了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50kb,覆盖了32.4倍基因组。并且插入片段均具有9～15个NotI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。更多还原

【Abstract】 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 ×haploid genome equivalents.And 9~15 Not I restriction site in the insert fragments,which is consistent with the features of Streptomyces genome.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.更多还原