地中海拟无枝菌酸菌U-32硝酸还原酶的基因克隆

Nitrate Reductase Gene Cloning of Amycolatopsis mediterranei U-32

Southern杂交分析表明在地中海拟无枝菌酸菌U-32染色体DNA和黑曲霉niaD(硝酸还原酶基因)之间存在着明显的同源性。利用异源niaD探针从地中海拟无枝菌酸菌U-32基因文库中筛选得到一个能与niaD杂交的5.0kb的PstⅠ片段。该片段经同位素标记后能与地中海拟无枝菌酸菌U-32染色体上一个相同的PstⅠ片段杂交,位于这一片段上的2.1kb SmaⅠ-EcoR Ⅴ片段只能与以硝酸盐为唯一氮源的总RNA杂交,而不能与相同条件下以铵盐为唯一氮源的总RNA杂交,这些结果表明,所克隆到的5.0kb PstⅠDNA片段含有地中海拟无枝菌酸菌U-32的硝酸还原酶基因。这是好氧细菌硝酸还原酶基因克隆的首次报道。由该酶蛋白分子量推测,其结构基因大小在1.5kb左右,进一步的杂交分析发现在5.0kb的PstⅠ片段中含有完整的NR基因。用20种限制酶对重组质粒pJL1进行了限制酶酶谱的构建,发现有10种酶在pJL1外源片段上无切点,6种酶为单切点,EcoRⅠ与SmaⅠ各有两个切点。

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.