摩拉水牛及德宏水牛瘤胃产甲烷菌多样性比较

16S rDNA Library-based Analysis of Ruminal Archaeal Diversity in Murrah Buffaloes and Dehong Buffaloes

本试验旨在比较分析摩拉水牛及德宏水牛瘤胃液中产甲烷菌的多样性。选取健康雌性摩拉水牛及德宏水牛各3头,采用口腔导管法收集瘤胃液,酚—氯仿—异戊醇抽提法提取瘤胃液总DNA,用产甲烷菌特异性引物Met86F/Met1340R扩增产甲烷菌16SrDNA,构建16SrDNA基因克隆文库。摩拉水牛及德宏水牛各获得96个16SrDNA基因序列,均归类于Methanobacteriales目,其中德宏水牛有82个序列(18个OTUs)与已知菌的16SrDNA序列相似性≥97%,占总序列的85.4%,有14个序列(9个OTUs)与已知菌16SrDNA序列相似性为89%~97%;摩拉水牛有94个序列(13个OTUs)与已知菌16SrDNA序列相似性≥97%,占总序列的97.9%,仅有2个序列(1个OTUs)与已知菌16SrDNA序列相似性为94%。系统进化树分析表明,所有序列分别聚集于两大分支上,其中德宏水牛有13个OTUs代表序列和摩拉水牛7个OTUs代表序列聚集在进化树顶端的同一分支上,且在系统发育距离上与Methanobacteriales目中任何已知相似序列都相隔较远。德宏水牛瘤胃中的SGMT簇序列和RO簇序列所占总序列比列分别为83.3%、9.4%,摩拉水牛瘤胃中的SGMT簇序列和RO簇序列所占总序列的比列分别为51.0%、9.4%。由此可见,摩拉水牛及德宏水牛瘤胃产甲烷菌序列以Methanobacteriales目为主,其中德宏水牛拥有更多未知的产甲烷菌序列;德宏水牛瘤胃中的SGMT簇产甲烷菌序列比例要高于摩拉水牛。

The objective of this study was to investigate the diversity of ruminal archaea in Murrah buffaloes and Dehong buffaloes through 16S rDNA library-based analysis.Three female Murrah buffaloes and three female Dehong buffalo were used in this study and rumen liquid were taken by stomach tube.Total DNA were extracted by phenol-chloroform-isopentanol extraction method.Primers of Met86 F/Met1340 Rwere used to amplify the 16S rDNA of archaea for the construction of library.The results showed that 96 sequences were obtained from each library of Murrah buffaloes and Dehong buffaloes.Based the 97% similarity,sequences of Murrah buffaloes and Dehong buffaloes were assigned to 27 and 14 OTUs,respectively.For Dehong buffaloes,82 sequences showed≥97%similarity to known species,14 sequences had the similarity to known species in the range of 89%to 97%.For Murrah buffaloes,94 sequences showed≥97%similarity toknown species,only two sequences had 94%（97%）similarity to known species.Phylogenetic analysis indicated that these sequences were assigned into two large clades.There were 13 OTUs representative sequences from Dehong buffaloes and 7 OTUs representative sequences from Murrah buffaloes assigned into the same clade on the top of the phylogenetic tree and far from all known species.They might be represented new genus or species in Methanobacteriales order in the rumen of Murrah buffaloes and Dehong buffaloes.The SGMT clade sequence and RO clade sequence percentage in Dehong buffaloes and Murrah buffaloes were 83.3% and 9.4%,51.0% and 9.4%,respectively.It could be concluded that Methanobacteriales order was the dominant archaea in the rumen of Murrah buffaloes and Dehong buffaloes.There were more unknown methanogen sequences and SGMT clade sequences in Dehong buffaloes than Murrah buffaloes under the same diet.