Nitrate Reductase Gene Cloning of Amycolatopsis mediterranei U-32
地中海拟无枝菌酸菌U-32硝酸还原酶的基因克隆

Southern blot analysis showed great homology existed between niaD (NR gene) of Aspergillus nidulans and A. mediterranei U-32 chromosome DNA. A 5.0 kb PstI fragment from A. mediterranei U-32 complementary to A. nidulans niaD gene was cloned in E. colt NM522 using niaD as a probe. An identical DNA band was observed through back-hybridization of the cloned DNA fragment to chromosome DNA of A. mediterranei U-32. Its 2.1kb Sma I -EcoR V fragment can only hybridize with total RNA from nitrate-cultured mycelium. These data suggested that the cloned DNA fragment contains NR gene of A. mediterranei U-32. This is the first report on NR gene cloning from aerobic bacteria. It was deduced from molecular weigh of NR that its gene sequence is about 1. 5kb in size. Further hybridization analysis indicated that the cloned DNA fragment covers the full-length NR gene of A. mediterranei U-32. We also constructed the physical map of the recombinant plasmid pJL1-with various restriction endonuclease, among them ten with no restriction site, six with unique site and two with double sites on the insert.