武夷菌素产生菌Fosmid文库的构建及文库探针的获得  

Construction of a Fosmid Library of Wuyiencin' s Producing Strain and Obtaining of the Library Probe

作　　者：檀贝贝[1] 孙蕾[1] 张克诚[1] 杨振娟[1] 武哲[1] 张俊[1] 

机构地区：[1]中国农业科学院植物保护研究所植物病虫害生物学国家重点实验室,北京100193

出　　处：《生物技术通报》2011年第6期116-121,共6页Biotechnology Bulletin

基　　金：公益性行业科研专项资金项目(201103002);中国农业科学院植物保护研究所中央级公益性科研院所基本科研业务费专项资金项目(161014201 1004)

摘　　要：以不吸水链霉菌武夷变种CK-15为材料，提取基因组DNA，经Sau3AI部分酶切后回收35—40kb之间的片段。连接到pCC1FOS载体上，经过包装转染涂布后构建得到了CK-15基因组的Fosmid文库，文库的滴度为8．8×10^5CFU／mL。随机挑取16个阳性克隆，经EcoR I和Hind Ⅲ双酶切电泳分析，样品插入片段平均长度大于35kh，插入率为100％，符合构建文库的要求。根据多氧霉素、尼克霉素生物合成基因及大环内酯类聚酮合成酶基因（PKS）设计特异性引物，以基因组为模板进行PCR扩增，筛选特异性引物探针。结果用大环内酯类聚酮合成酶基因设计引物扩增出1693bp的片段经Blast比对其与大环内酯类抗生素生物合成基因相似性为95％以上。武夷菌素产生菌CK-15 Fosmid文库的构建及文库探针的获得，为武夷菌素生物合成基因的克隆奠定了基础。Streptomyces ahygroscopicus var. wuyiensis was taken as the material. Its total genome was digested partially by enzyme Sau 3A I , and 35 -45 kb fragments was extracted. Then the extracted fragments were ligated to vector pCCI FOS. After packaged and transfected,the Fosmid library of Wuyiencin＇ s producing strain was constructed. The titer of the Fosmid library was 8.8 ×10s CFU/mL. We picked 16 positive clones and digested with EcoR I and Hind HI. As a result,the electrophoresis showed that the average sizes of the inserting DNA ligated to the vectors were greater than 35 kb,and the inserting rate was 100%. According to the biosynthetic gene cluster of Nikkomycin, polyoxin and macrolides antibiotics, we designed some specific primers to PCR using genome DNA as template for probe screening. The result showed that the primes designed by using PKS amplified DNA fragment about 1 693 bp which had high simi- larity with other biosynthetic genes of macrolides antibiotics. Construction of a Fosmid library of Wuyiencin＇ s producing strain and obtaining of the library probe will make a foundation for cloning of biosynthetic genes of Wuyiencin.

关 键 词：武夷菌素 基因组 FOSMID文库 文库探针 

分 类 号：S482.2[农业科学—农药学]