环境样品中亚硝酸细菌amoA基因的克隆与测序

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

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。

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.