利用16S rRNA基因克隆文库技术分析德昌水牛瘤胃产甲烷菌的多样性

16S rRNA library-based analysis of ruminal methanogens diversity in Dechang water buffalo

选取3头5岁左右的健康雌性德昌水牛作为试验动物,采用机械破壁法提取瘤胃内容物总DNA,用产甲烷菌特异性引物Met86F/Met1340R扩增16S rRNA基因,构建16S rRNA基因克隆文库,分析德昌水牛瘤胃产甲烷菌区系组成。结果表明：试验共获得99个16S rRNA基因序列,RDP分析表明94.2%的序列为甲烷短杆菌属（Methanobrevibacter）16S rRNA序列,按照97%的相似性划分为19个分类操作单元,其中96个序列（17个OTUs）占总序列的97.0%,与已知细菌的16S rRNA序列的相似性≥97%；3个序列（2个OTUs）占总序列的3.0%,与已知细菌16S rRNA序列的相似性为90%～97%；系统发育树分析表明,SGMT簇序列和RO簇序列所占总序列的比例分别为75.8%、1.0%,部分序列与Methanobrevibacter中任何已知相似序列都相隔较远,它们可能代表Methanobrevibacter中新的种。以上结果表明,德昌水牛瘤胃产甲烷菌以Methanobrevibacter产甲烷菌为优势菌群,其中有许多未培养的产甲烷菌需进一步分离培养,并对其功能进行分析。

Three femal Dechang water buffaloes were used in this study. Total DNA were extracted by bead-beating method. Primers of Met86F/Met1340 R were used to amplify the 16 S rRNA of archaea for the construction of library. Results showed that 99 sequences were obtained. RDP analysis showed that 94.2% sequences are classified to genus Methanobrevibacter. Based the 97% similarity, these sequences were assigned to 19 OTUs. Ninety-six sequences showed ≥97% sequence similarity to known species, three sequences had sequence similarity to known species in the range of 90% to 97%. Phylogenetic analysis indicated that 75.8% archaeal sequences in the rumen of Dechang water buffaloes belonged to SGMT clade, only 1.0% sequences belonged to RO clade. There are some sequences far from all known species on the phylogenetic tree. They may represented new species in Methanobrevibacter genu in the rumen of Dechang water buffaloes. It can be concluded that Methanobrevibacter genu is the dominant archaea in the rumen of Dechang water buffaloes. Further study should be done for the isolation of uncutured methanogens and their function analysis.