天蓝淡红链霉菌SIPI1482中柔红霉素C-14羟化酶基因的克隆和序列分析

Cloning and sequencing of daunomycin C-14 hydroxylase gene from Streptomyces coeruleorubidusSIPI1482

根据 Gen Bank中公布的两种柔红霉素 C- 14羟化酶基因 (dox A)的序列 ,设计引物并通过 PCR扩增方法从天蓝淡红链霉菌 SIPI14 82中扩增出约 1.4 kb的目的片段 ,其中包括了长度为 12 6 9bp的 dox A全长序列 ,而同时从波赛链霉菌 SIPI4 0 14 1中没有扩增出基因片段。将扩增出的 dox A基因和载体质粒 p Blue-script KS连接后构建了质粒 p YG5 10 ,通过 PCR扩增、酶切鉴别及测序分析发现 ,天蓝淡红链霉菌 SIPI14 82来源的 dox A基因和链霉菌 C5来源的序列完全相同。

A pair of primers were designed based on two C-14 hydroxylase gene (doxA) sequences published in GenBank and a DNA fragment of approximate 1.4kb was amplified from Streptomyces coeruleorubidusSIPI1482 by PCR method. However, none of DNA fragment was amplified from S.peucetiusSIPI40141. The amplified doxAgene was ligated into vector pBluescript KS and the recombinant plasmid pYG510 was identified for the presence of doxAgene by both PCR amplification and enzyme digestion. Sequencing analysis indicated that the sequence of the cloned doxAgene from S.coeruleorubidusSIPI1482 is the same as the published doxAgene from Streptomycessp. C5.