黑暗链霉菌安普霉素生物合成基因aprG的克隆和功能研究

从安普霉素产生菌S．tenebrarius H6总DNA中已克隆得到8．2kb的安普霉素抗性基因连锁片段。对距离抗性基因下游4．5kb的SacI-Sau3AI片段进行测序分析，发现了一个新的891bp开放阅读框架。推测其编码为296个氨基酸的蛋白质。经BLASTX程序分析，没有发现与之同源的基因，是一个新基因，该基因命名为aprG。构建基因阻断穿梭载体pSPU58，经接合转移导入S．tenebrarius H6，筛选单交换阻断变株，并用Southern blot验证阻断变株的aprG已经被破坏。经发酵分析，阻断变株不再合成安普霉素，表明aprG与安普霉素生物合成直接相关。

Cloning and function analysis of aprG invloved in apramycin biosynthesis in Streptonyces tenebrarius H6

The 8 2kb DNA fragment containing apramycin resistant gene ( aprB ) has been cloned from S.tenebrarius H6 total DNA. The Sac I Sau 3AI DNA fragment, located at 4 5kb downstream of the aprB gene, was sequenced. Analysis of the nucleotide sequence revealed that it contained an ORF composed of 891bp encoding a 296 amino acid hypothetical protein. By analysing with BLASTX, no homology was found, so that the cloned gene was supposed to be an unknown new gene, designated as aprG . The plasmid pSPU58 for gene disruption was constructed and transfered into S.tenebrarius H6 by conjugation. The southern blot experiment showed that the aprG was disrupted correctly. The disruptant no longer produced apramycin, so we can concluded that aprG was closely related to apramycin biosynthesis.