亚硝酸细菌amoA基因的克隆、测序与表达  

CLONING, SEQUENCING AND EXPRESSION OF amoA GENE OF NITROSBACTERIA

作　　者：李君文[1] 周娟[1] 王新为[1] 郑金来[1] 古长庆[1] 宋农[1] 金敏[1] 晁福寰[1] 

机构地区：[1]军事医学科学院卫生学环境医学研究所,天津300050

出　　处：《应用与环境生物学报》2004年第3期345-348,共4页Chinese Journal of Applied and Environmental Biology

基　　金：国家"863"项目(No.2001AA214191);天津市科技攻关重大项目(No.023180711)资助~~

摘　　要：采用PCR技术对亚硝酸细菌的特异性amoA基因进行克隆、测序 ,以确定其准确性 ;然后采用基因重组技术构建亚硝酸细菌的基因工程菌 ,并进行鉴定与功能初步评价 .结果表明 ,克隆得到的 4种亚硝酸细菌的amoA基因 ,与标准菌株Nitrosomonassp.GH2 2的amoA基因同源性达到 98%～ 99% ;构建了一种含有特异性amoA基因的大肠杆菌基因工程菌株 ,并采用直接比色法对重组细菌的氨氧化活性进行测定 ,发现基因工程菌的氧化氨氮速率明显高于分离的野生菌株 .研究表明 ,利用基因重组技术对亚硝酸细菌等自养菌进行改造 ,构建高效基因工程菌在水污染治理领域具有可行性 .图 6表 1参The specific amoA (ammonia monooxygenase)gene was cloned and sequenced, then the recombination bacteria was established and the recombination bacteria activity of ammonia oxidation was evaluated. The whole long gene amoA of four Nitrosomonas sp. was amplified and sequenced, and blasted in Genbank through Internet, respectively. The results showed that all amplified DNA fragments were in high homology with the amoA gene of Nitrosomonas sp. GH22 (98%～99%). amoA gene was ligated into PQE30 plasmid and then transformed into E.coli M15. After blue white selection, PCR identification, and digestion with Bam HⅠand Hin dⅢ proved that expression system of amoA gene was successfully constituted. The ability of the recombination bacteria to oxidize ammonia was evaluated by measuring the concentrations of ammonia in the incubation medium. The results showed that the ammonia oxidation activity of one recombination clone was higher than that of the wild strain. The results suggest it be possible to establish the recombination bacteria of Nitrosomonas to oxidize the ammonia in wastewater. Fig 6, Tab 1, Ref 12

关 键 词：亚硝酸细菌 基因重组 氨氧化 

分 类 号：X172[环境科学与工程—环境科学]