转dct结构基因固氮斯氏假单胞菌工程菌株构建及其特性研究  

Construction of Recombinant Strains of Pseudomonas stutzeri A1501 with an Extra Copy of dctPQM and Study on Their Physical Characterization

作　　者：闫春玲[1] 林敏[1] 徐玉泉[1] 平淑珍[1] 陈明[1] 张保明[2] 

机构地区：[1]中国农业科学院生物技术研究所,北京100081 [2]中国农业科学院作物育种栽培研究所,北京100081

出　　处：《高技术通讯》2003年第10期36-40,共5页Chinese High Technology Letters

基　　金：86 3计划 (2 0 0 1AA2 14 0 2 1) ;国家自然科学基金 (395 80 0 0 1)资助项目

摘　　要：固氮斯氏假单胞菌 (Pseudomonasstutzeri)A15 0 1是一种定殖于水稻根部的联合固氮菌。将含有A15 0 1四碳二羧酸转运系统 (Dct transportsystem)结构基因dctPQM的亚克隆大片段克隆到具有广泛宿主范围转移特性的质粒载体 pSZ2 1上。通过三亲接合试验 ,重组质粒 pSZY6中所携带的dctPQM基因随机转座插入到菌株A15 0 1的染色体基因组中 ,构建了含有额外拷贝dct结构基因的遗传工程菌株。在A15卡那霉素 (5 0 μg/mol)平板上 ,共筛选到约 6 0株转接合子。其中工程菌株A 136、A 115和A 14 2在以不同的四碳二羧酸如琥珀酸、延胡索酸和苹果酸 ,以及乳酸为唯一碳源生长时 ,表现了较高的固氮活性 ,其固氮水平明显高于野生型菌株A15 0 1。P.stutzeri A1501 is an associative nitrogen fixation bacteria, which colonizes the rhizosphere of many important cereals, such as paddy, and spreads extensively in China. Dct system is a trans membrane transport system that transports periplasmic C4 dicarboxylic acids into cytoplasm. Bacteria utilize these carbon sources for basic metabolism and nitrogen fixation. The fragment containing structural gene(s) dctPQM was cloned into a broad host mobilization vector pSZ21 and the resultant plasmid named pSZY6. By tri parental mating assay, the recombinant plasmid was transferred into A1501, and Tn5 carrying dctPQM of pSZY6 inserted into the genome of A1501 randomly. So the recombinant strains with an extra copy of dct structural gene(s) have been constructed. Almost 60 conjugants were obtained, 3 of them expressed high activity of nitrogen fixation when c4 dicarboxylates succinate, fumarate, malate and lactic acid as the sole carbon source. Activity of nitrogen fixation of recombinant strains A 115, A 136 and A 142 are higher obviously than the wild type strain A1501.

关 键 词：固氮斯氏假单胞菌 水稻 染色体 四碳二羧酸运输系统 Dct系统 

分 类 号：Q789[生物学—分子生物学]