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环境样品中亚硝酸细菌amoA基因的克隆与测序

周娟 李君文 郑金来 王新为 宋农 古长庆

周娟, 李君文, 郑金来, 王新为, 宋农, 古长庆. 环境样品中亚硝酸细菌amoA基因的克隆与测序[J]. 环境科学研究, 2004, 17(2): 77-80.
引用本文: 周娟, 李君文, 郑金来, 王新为, 宋农, 古长庆. 环境样品中亚硝酸细菌amoA基因的克隆与测序[J]. 环境科学研究, 2004, 17(2): 77-80.
ZHOU Juan, LI Jun-wen, ZHENG Jin-lai, WANG Xin-wei, SONG Nong, GU Chang-qing. Cloning and Sequencing of Ammonia-Oxidizing Bacteria amoA Gene from Environmental Samples[J]. Research of Environmental Sciences, 2004, 17(2): 77-80.
Citation: ZHOU Juan, LI Jun-wen, ZHENG Jin-lai, WANG Xin-wei, SONG Nong, GU Chang-qing. Cloning and Sequencing of Ammonia-Oxidizing Bacteria amoA Gene from Environmental Samples[J]. Research of Environmental Sciences, 2004, 17(2): 77-80.

环境样品中亚硝酸细菌amoA基因的克隆与测序

基金项目: 国家"836"计划项目(2001AA214191,2002AA601240);天津市科技攻关重大项目(0231807111)

Cloning and Sequencing of Ammonia-Oxidizing Bacteria amoA Gene from Environmental Samples

  • 摘要: 对从环境样品中分离的亚硝酸细菌(Ammonia oxidizingbacteria)amoA基因进行克隆与测序,为构建基因工程菌打下基础。采用亚硝酸细菌选择性培养基,从4个不同的畜牧养殖污水处理厂采集的样品(分别编号为1,2,3,4)在室温下富集培养2个月后,采取酚氯仿抽提的方法提取DNA。根据已报道的亚硝化单胞菌(Nitrosomonassp.)amoA基因序列,设计引物AMOB AMOE,并在AMOB,AMOE的5′-端分别加上了BamHⅠ和HindⅢ的限制性酶切位点,以利于进一步酶切和克隆。用AMOB AMOE对4种样品的DNA进行PCR扩增,PCR产物进行琼脂糖凝胶电泳分析。结果表明,4种样品中1号和3号样品扩增得到预期长度的DNA片段,2号和4号样品扩增没有得到预期片段。回收纯化PCR产物与pGEM-T载体连接,构建amoA基因测序载体,并转化E.coliM15。测序结果提交GenBank进行Blast分析。结果显示,扩增得到的DNA片段均与Nitrosomonassp.GH22的amoA基因有99 7%的同源性,可从环境中分离的亚硝酸细菌中克隆出amoA基因。

     

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  • 收稿日期:  2003-05-06
  • 刊出日期:  2004-04-25

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