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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

黄黎, 梁建光, 吴永强

中科院上海植物生理研究所.上海 200032

摘要

以质粒pKT230为载体,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DH5#alpha#/pHR11、DH5#alpha#/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在催娩克氏杆菌中传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_(2)诱导接合子NGH999和NGH982吸氢酶活性高表达,同时固氮效率和固氮酶活性也高。低碳源浓度更有利于接合子吸氢酶活性的表达和固氮效率的提高。接合子NGH982具有耐铵的基因,因此其固氮酶活性的表达有部分去铵阻遏的作用。

关键词： 催娩克氏杆菌; 质粒稳定性; 吸氢酶活性; 吸氢酶基因

The stabilization of plasmid carrying hup genes and the expression of H_(2)-uptake hydrogenase in Klebsiella oxytoca

Huang Li, Liang Jianguang, Wu Yongqiang

Shanghai Institute of Plant Physiology,Chinese Academy of Sciences.Shanghai 200032

Abstract

The resulting transconjugants were analyzed for plasmid stability. Plasmid pKH1 was found to have high stability in its host K. oxytoca. High levels of H_(2)-uptake hydrogenase activity and nitrogen-fixing efficiency relative to the wild type were observed in the two transconjugants NGH999 and NGH982. The increase was more remarkable in a medium with low sucrose content. Transconjugant NGH982 showed partially derepressional expression of nitrogenase in the presence of ammonium.

Key words: Klebsiella oxytoca;Stabilization of Alasmid;H_(2)-uptake hydrogenase activity;Hup gene

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引用本文：

黄黎, 梁建光, 吴永强. 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[J]. 生理学报 2001; 53 (1): .

Huang Li, Liang Jianguang, Wu Yongqiang. The stabilization of plasmid carrying hup genes and the expression of H_(2)-uptake hydrogenase in Klebsiella oxytoca. Acta Physiol Sin 2001; 53 (1): (in Chinese with English abstract).