| 摘要: |
| 利用脲酶保守序列ureC引物和细菌16S rDNA通用引物PCR扩增并测序,揭示奶牛瘤胃尿素分解菌的多样性。提取本研究室前期鉴定的16个脲酶克隆的质粒,利用脲酶保守序列ureC引物和细菌16S rDNA通用引物进行PCR扩增,将PCR产物连接到pMD19-T载体,转化E.coli JM109,挑取阳性克隆测序。利用Blast程序将序列与GenBank和RDP数据库进行比对,并使用Mega 4.0软件构建系统发育树。4个脲酶克隆含有脲酶保守序列ureC,系统发育树分析表明,U1、U5、U13和U15分别属于Firmicutes、ε -Proteobacteria、β -Proteobacteria和Actinobacteria;7个脲酶克隆含有16S rDNA,经过建树分析,U1、U3、U7、U10、U11、U12和U14分别属于Staphylococcus、Shigella、Bacillus、Acinetobacter、Achromobacter、未培养微生物和Bacillales。实验结果显示,奶牛瘤胃微生物脲酶基因和尿素分解菌具有多样性的特点。 |
| 关键词: 脲酶 尿素分解菌 多样性 宏基因组学 16S rDNA ureC |
| DOI：10.11841/j.issn.1007-4333.2010.01.010 |
| 投稿时间：2009-04-15 |
| 基金项目:北京市自然科学基金资助项目(6092017);动物营养学国家重点实验室自主研究课题(2004DA125184(团)0801)第一作者:赵圣国,硕士研究生,E-mail:zhaoshengguo1984@163.com通讯作者:王加启,研究员,博士生导师,主要从事反刍动物营养和牛奶质量改良研究,E-mail:wang-jia-qi@263.net |
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| Analysis for the diversity of ureolytic bacterium from dairy rumen based on metagenomics |
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ZHAO Sheng-guo, WANG Jia-qi, LIU Kai-lang, LI Dan, YU Ping
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| (State Key Laboratory of Animal Nutrition/Institute of Animal Science/Chinese Academy of Agricultural Sciences, Beijing 100193,China) |
| Abstract: |
| The purpose of this study was to reveal the phylogenetic diversity of ureolytic bacterium from dairy rumen microbial by PCR using urease conserved ureC degenerate primer and bacterial 16S rDNA universal primer and sequencing. All of the previously identified plasmids for the sixteen urease positive clones which were identified previously were exacted. Polymerase chain reaction degenerate primers based on the ureC gene which was conserved in ureolytic bacterium were designed and used to amplify the ureC genes from the extracted plasmids. The universal 16S bacterial primers were used to amplify the 16S rDNA gene of positive clones. PCR products were purified and ligated to pMD19-T vector. Transformants got by transforming ligation product into E.coli JM109 were sequenced. The sequences were analyzed by Blast on GenBank and RDP, and the phylogenetic trees were built by Mega 4.0. In results, four clones (U1, U5, U13 and U15) containing ureC genes were obtained. The phylogenetic tree of ureC revealed that all four clones were clustered into several bacterial phylums. U1, U5, U13 and U15 belonged to Firmicutes, ε-Proteobacteria, β-Proteobacteria and Actinobacteria, respectively. U1, U3 , U7, U10, U11, U12 and U14 which contained the 16S rDNA sequence had the most identity to Staphylococcus,Shigella,Bacillus,Acinetobacter,Achromobacter,uncultured bacterium and Bacillale. The diversity of ureC and 16S rDNA revealed the diversity of ureolytic bacterial in dairy rumen. |
| Key words: urease ureolytic bacterium diversity metagenome 16S rDNA ureC |
