含hup基因的质粒在催娩克氏杆菌中的稳定性及吸氢酶的表达

以质粒pKT230为栽体，亚克隆大豆根瘤菌吸氢酶结构基因(hupSL)片段，构建成嵌合质粒pKH1。将质粒分配系统基因(parDE)片段和吸氢酶结构基因(hup)片段插入载体质粒pRK415，构建成质粒pRKBH。质粒pKH1、pRKBH和载体pRK415经转化和三亲本杂交，得到DH5α/pHR11、DH5α/pRKBH、E1201/pKH1、NG13/pKH1(NGH999)、NG1390/pRK415、NG1390/pHR11、NG1390/pRKBH和NG1390/pKH1(NGH982)等接合子。稳定性分析发现，质粒pKH1在催娩克氏杆茵中传80代后仍有92％以上的菌株含此质粒，说明质粒pKH1有较高的稳定性。吸氢酶活性分析表明，H

The Stabilization of Plasmid Carrying hup Genes and the Expression of H2 -uptake Hydrogenase in Klebsiella oxytoca

Structural genes of hup, hupSL, iso lated from Bradyrhizobiumjaponicum in a 5.9-kb Hind Ⅲ fragment, were subcloned into the vector plasmid pKT230, and a chimeric plasmid pKH1 was obtained ( Fig. 1 ). Partitioning genes parDE and hupSL were subcloned into vector plasmid pRK415, resulting in plasmid pRKBH (Fig. 2). Then the plasmids were mobilized into strains of E. coli DHS, Enterobacter cloacae El201, K. oxyoca NG13 (wild type)and NG1390 (nifAc genotype) by triparentalmating using the helper pRK2013, and DH5α/pHRll, DH5α/pRKBH, E1201/pKH1, NG13/pKH1(NGH999) ,NG1390/pRK415, NG1390/pHRll, NG1390/pRKBH and NG1390/pKH1( NGH982 ) were obtained. The resulting transconjugants were analyzed for plasmid stability (Fig. 3 ). Plasmid pKH1 was found to have high stability in its host K. oxytoca. High levels of H2-uptake hydrogenase activity and nitrogen-fixing efficiency relative to the wild type were observed in the two transconjugants NGH999 and NGH982 (Table 2 and Fig. 4).The increase was more remarkable in a medium with low sucrose content (Fig. 4). Transconjugant NGH982 showed partially derepressional expression of nitrogenase in the presence of ammonium ( Fig. 5 ).