硫霉素产生菌卡特利链霉菌A520基因文库的构建

Construction of genomic library from Streptomyces cattleya A520

以大肠杆菌／链霉菌穿梭柯斯质粒ｐＫＣ５０５为载体，构建了卡特利链霉菌（Ｓｔｒｅｐｔｏｍｙｃｅｓｃａｔｌｅｙａ）Ａ５２０在大肠杆菌ＤＨ１的基因文库，重组质粒插入外源片段的概率在９５％以上，插入外源片段的大小为２０～３０ｋｂ。以链霉菌染色体分子量为１０４ｋｂ计算，由４０００个转化子构成的基因文库可以概括卡特利链霉菌Ａ５２０约９９％的基因组。用已经克隆的硫霉素环化酶基因上游的１．５ｋｂＤＮＡ片段作为探针杂交，从基因文库得到３２个阳性克隆。用利波曼链霉菌（Ｓ．ｌｉｐｍａｎｉ）中的ＩＰＮＳ基因作为探针杂交，从基因文库得到１个阳性克隆ｐＫＷ２０１／ＤＨ１，并为Ｓｏｕｔｈｅｒｎ杂交进一步证实杂交片段在ＢａｍＨⅠ２．７ｋｂ片段上。用来自带小棒链霉菌（Ｓ．ｃｌａｖｕｌｉｇｅｒｕｓ）的克拉维酸生物合成途径中的环化酶基因ｃｓ２作为探针杂交进行筛选，得到１个阳性克隆ｐＫＷ３０１／ＤＨ１，说明所构建的基因文库比较完整。

The genomic library from the S.cattleya A520 was established in E.coli DH1 by using pKC505, a E.coli/Streptomyces cosmid vector, in vitro packaging. Foreign DNA fragments and the frequency of insertion in recombinated plasmid were 23￣30kb in size and over 95% respectively. Assuming that the Streptomyces genome is about 10 4 kb in size, the probability of finding a specific gene from the library composed of 4000 colones is approx 99%. A 1.5 kb upstream DNA fragment of the thienamycin cyclase gene isolated from the S.cattleya A520, an IPNS gene from the S.lipmanii and a cs2, a clavulanic acid cyclase gene, from the S.clavuligerus were used as probes; 32, 1 and 1 positive clones were isolated from the library, respectively. This result proved that the constructed genomic library from the S.cattleya A520 was complete. The relationship among three kinds of clones isolated by different probes and the genes related to thienamycin biosynthesis are being studied further.