Two exchange arms, which were respectively located upstream and downstream of gene ctcF encoding methylase of the C6 position of chlortetracycline, were amplified by PCR from Streptomyces aweofaciens J13 genomic DNA. The marker gene neo and constitutive promoter ermE# were then inserted between the arms. With these fragments, we constructed pSET152-derived vector pNEO152. The recombinant vector was transformed to Escherichia col ET12567 (pUZ8002) , and then transferred to S. Aureofaciens J13 by conjugation. The positive transfonnants were selected on MS plate. After PCR identification, it was confirmed that pFD106 was integrated into the chromosome.%以金色链霉菌J13基因组DNA为模板,扩增金霉素C6甲基化酶基因ctcF上下游序列作为两端同源交换臂,在两臂之间添加抗性筛选标记neo基因,并在该基因上游加入组成型强启动子ermE*,以强化筛选标记.将该外源DNA序列插入到质粒pSET152,构建重组质粒pFD106.转化大肠杆菌ET12567后,经接合转移导入金色链霉菌J13中,MS平板上筛选阳性接合子.PCR检测表明,重组质粒已整合到染色体DNA上.
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