柔红霉素生物合成基因簇的克隆及其基因打靶研究  

Clonging of Gene Cluster for Daunorubicin Biosynthesis and the PCR-Targeting Study

作　　者：袁天杰[1,2] 谢丽萍[1,2] 朱宝泉[1,2] 胡又佳[1,2] 

机构地区：[1]中国医药工业研究总院上海医药工业研究院 [2]上海欣生源药业有限公司,上海200437

出　　处：《中国医药工业杂志》2014年第12期1128-1134,共7页Chinese Journal of Pharmaceuticals

基　　金：国家“重大新药创制”科技重大专项(2011ZX09203)

摘　　要：本研究通过构建柔红霉素产生菌天蓝淡红链霉菌Streptomyces coeruleorubidus DM的基因组文库,获得2 230个重组克隆。利用菌落原位杂交和PCR筛选获得黏粒Cosmid 8-22,其插入片段大小为41 431 bp,含36个完整的开放阅读框,基本包含了完整的柔红霉素生物合成基因簇。利用黏粒Cosmid 8-22成功构建了基因打靶系统,同时敲除部分dau W和完整的dnr X基因。HPLC结果显示,敲除突变株的柔红霉素产量较出发菌提高了3~4倍。Genome of Streptomyces coeruleorubidus DM, the producing strain of daunorubicin, was used to construct the gene library, and a total of 2 230 recombinants were obtained. The recombinant Cosmid 8-22 was obtained by colony in situ hybridization and PCR amplification. The insert fragment of Cosmid 8-22 was 41 431 bp in length and harbored 36 intact ORFs which almostly contained the complete DNR biosynthetic gene cluster. The Cosmid 8-22 was used to establish the PCR-targeting system, which could disrupt partial dau W and complete dnr X at the same time. The HPLC results showed that the DNR production of mutant strain was increased significantly, which was nearly about 3- 4 folds higher than the parental strain.

关 键 词：柔红霉素 基因组文库 基因打靶 基因敲除 

分 类 号：Q78[生物学—分子生物学]