Phylogenetic analysis of rumen bacterial 16s rRNA gene sequences of gayal

Chinese. 【目的】研究大额牛（Bos frontalis）瘤胃细菌16S rRNA基因序列,为揭示大额牛瘤胃细菌组成的多样性以及开发这一珍稀生物资源提供分子生物学依据。【方法】采用细菌通用引物F27和R1492对目标基因进行PCR扩增,克隆、测序后利用Neighbor-joining方法构建系统进化树。【结果】共获得147个16S rRNA基因序列,在GenBank登录号为DQ673466～DQ673612。按照97%的相似性标准,这些序列被分为86个操作分类单元。有16个序列与已培养菌的序列相似性≥97%,占总序列的10.9%;另有22个序列与已培养菌的序列相似性为90%～97%,占总序列的15%;剩余109个序列为未培养、鉴定菌,所占比例高达74.1%。系统进化分析显示,大额牛瘤胃细菌主要分布于低G＋C含量细菌门（57.1%）和噬纤维菌/屈扰杆菌/拟杆菌门（42.2%）,仅有一个序列位于螺旋体门。【结论】本试验获得了大额牛瘤胃细菌16S rRNA基因序列,为进一步研究大额牛瘤胃微生态系统组成及其与饲料消化之间的关系奠定了基础。

English. [Objective] The objective of present work is to reveal the rumen bacterial 16S rRNA gene sequences ofgayal （Bos frontalis）, and to provide molecular basis for its bacterial diversity and more exploitation. [Method] Universal primers ofF27 and R1492 were used for PCR amplification of the target sequences, which were conducted neighbor-joining phylogenetic analysis after cloning and sequencing. [Result] A total of 147 clones were obtained and submitted to GenBank under accession numbers of DQ673466-DQ673612, which were referred as 86 Operational Taxonomy Units （OTUs） according to the similarity standard of 97%. Sixteen sequences were affiliated to the cultured rumen bacteria, accounting for 10.9% of the total clones. For 15% （22 sequences） of the sequences, the similarity with the database was in the range of 90.0%-97.0%. While for the remaining 74.1%, the similarity was less than 90%. Based on the phylogenetic analysis, the majority of the cloned sequences fell into the phyla of low G＋C subdivision （57.1%） and Cytophaga-Flexibacter-Bacteroides （CFB,42.2%）, respectively, and only one clone was related to the phylum of Spirochaetes. [ Conclusion] Rumen bacterial 16S rRNA gene sequences of gayal （Bos frontalis） were successfully obtained and analyzed phylogenetically, which would promote the studies of the rumen microbial ecosystem of the endangered cattle.