不吸水链霉菌梧州新亚种基因组文库的构建

以不吸水链霉菌梧州新亚种为材料，提取的总基因组DNA经Sau3 AI不完全酶切。回收20-30kbDNA酶切片段，与经Hpal和BamHI酶切的粘粒载体pKC505进行连接，将连接产物用噬菌体包装蛋白包装。侵染大肠埃希菌DH5α。在舍有安普霉素的LB平板上培养，所得转化子数为12000个，构建成不吸水链霉菌梧州新亚种的基因组文库。以不吸水链霉菌染色体基因组大小为7Mb计算，概括了不吸水链霉菌梧州新亚种约99％的基因组，达到了建库要求的理论值。未扩增文库的滴度为1．2×10^8pfu／L，扩增文库的滴度为4．8×10^11pfu／L，包装效率为每微克DNA1．2×10^5个转化子。随机挑取菌落，提取重组质粒进行酶切电泳分析，均有外源片段插入。基因文库的构建为进一步深入研究梧宁霉素生物合成基因结构及功能奠定基础。

Construction of Gene Library of Streptomyces ahygroscopicus neosubsp.wuzhouensis

Streptomyces ahygroscopicus neosubsp, wuzhouensis （SANW） was taken as the material, its total genome was digested by enzyme Sau3AI partially, and 20 - 30 kb DNA fragments were recycled. The fragments were ligated with the Hpal and BamHI site of cosmid pKC505, and ligation product were packaged with bacteriophage packaging protein then transfectcd into E. coli DH5α in vitro. The number of transfectnants cultured in LB plate containing Amramycin was 12000, thus a SANW gene library was constructed. Regarded the chromosome size of SANW as about 7 Mb, this number generalized 99% genome of SANW approximately which reached the theoretical value requested to construct gene library. The titers of the unamplified and the amplified libraries were 1.2 ×10^8 pfu/L and 4.8×10^11 pfu/L respectively. The packaging efficiency was about 1.2 ×10^5 transfectants per ug DNA. Randomly select colonies then extract recombinant plasmid and carry out digestion and electrophoresis analysis; the results had proved that there were exogenous fragments inserted. The construction of the gene library had laid a foundation for further study on the structure and function of wuningmeisu biosynthetic gene.