| 环境样品中亚硝酸细菌amoA基因的克隆与测序 |
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| 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 |
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| 作者姓名： | 周娟 李君文 郑金来 王新为 宋农 古长庆 |
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| 作者单位： | 军事医学科学院,卫生学环境医学研究所,天津,300050;军事医学科学院,卫生学环境医学研究所,天津,300050;军事医学科学院,卫生学环境医学研究所,天津,300050;军事医学科学院,卫生学环境医学研究所,天津,300050;军事医学科学院,卫生学环境医学研究所,天津,300050;军事医学科学院,卫生学环境医学研究所,天津,300050 |
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| 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) |
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| 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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| 关 键 词： | 亚硝酸细菌 amoA基因 克隆 |
| 文章编号： | 1001-6929(2004)02-0077-04 |
| 收稿时间： | 2003-05-06 |
| 修稿时间： | 2003-05-06 |
Cloning and Sequencing of Ammonia-Oxidizing Bacteria amoA Gene from Environmental Samples |
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| ZHOU Juan,LI Jun-wen,ZHENG Jin-lai,WANG Xin-wei,SONG Nong and 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 |
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| Authors: | ZHOU Juan LI Jun-wen ZHENG Jin-lai WANG Xin-wei SONG Nong GU Chang-qing |
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| Affiliation: | Institute of Hygiene and Environmental Medicine,Academy of Military Medical Sciences,Tianjin 300050,China |
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| Abstract: | To provide basis of establishing gene-engineering bacteria,the ammonia-oxidizing bacteria amoA gene was cloned and sequenced.Samples(marked as 1,2,3,4) from four different environments were cultured in selective enrichment culture for ammonia-oxidizing bacteria.To amplify the whole long gene amoA,PCR primers were designed as AMOB/AMOE,and BamHⅠ and HindⅢ sites were added to the 5′-ends of each primer respectively to make the following steps easily.After amplification of the four samples,agarose gel electrophoresis of amplification product showed that samples 1 and 3 produced the expected product,but samples 2 and 4 did not.Retracted PCR products were ligated into pGEM-T vector to constitute sequencing vector.After being sequenced the sequences were blasted in GenBank through Internet.The results showed that amplified DNA fragments of sample 1 and 3 both have 99.7% homology with the amoA gene of Nitrosomonas sp.GH22.It could be concluded that the amoA gene was obtained from the exacted bacteria. |
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| Keywords: | ammonia-oxidizing bacteria amoA gene cloning |
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