尼里-拉菲水牛及海子水牛瘤胃产甲烷菌多样性比较

Comparative Analysis of Ruminal Archaeal Diversity Between Nili-Ravi Buffaloes and Haizi Buffaloes

试验旨在比较尼里-拉菲水牛及海子水牛瘤胃液中产甲烷菌的多样性,选取成年雌性尼里-拉菲水牛及海子水牛各3头作为试验动物,采用口腔导管法收集瘤胃液,提取瘤胃液总DNA,采用产甲烷菌特异性引物Met86F/Met1340R扩增16S rDNA,构建16S rDNA基因克隆文库。结果显示,从尼里-拉菲水牛和海子水牛中分别获得73、100条有效序列,均以Methanobrevibacter属为主。其中尼里-拉菲水牛有70条序列(17个OTUs)与已知菌的16S rDNA序列相似性≥97%,占总序列的95.89%,有3条序列(3个OTUs)与已知菌16S rDNA序列相似性处于93%~96%;海子水牛有70条序列(15个OTUs)与已知菌16S rDNA序列相似性≥97%,占总序列的70%,有30条序列(18个OTUs)与已培养菌16S rDNA序列相似性处于84%~96%。尼里-拉菲水牛和海子水牛瘤胃中的SGMT簇序列、RO簇序列所占总序列比例分别为76.71%、21.91%和76.00%、19.00%。系统进化树分析表明,所有序列分别聚集于两大分支上,其中海子水牛有16个OTUs代表序列和尼里-拉菲水牛8个OTUs代表序列聚集在进化树顶端的同一分支上,且系统发育距离与古菌中任何已知相似序列都相隔较远。综上所述,在本试验同等日粮条件下,尼里-拉菲水牛及海子水牛瘤胃内产甲烷菌序列均以Methanobrevibacter属为主,两水牛品种的SGMT簇序列比例基本一致,但尼里-拉菲水牛中的RO簇序列比例更高,海子水牛瘤胃内存在更多的未知产甲烷菌。

The objective of this study was to investigate the diversity of ruminal Archaea in Nili-Ravi buffaloes and Haizi buffaloes using 16S rDNA library-based analysis.Three female Nili-Ravi buffaloes and three female Haizi buffaloes were used in this study,and ruminal samples were taken by a stomach tube.Total DNA were extracted,and specific primers of Met86F/Met1340R were used to amplify 16S rDNA of methanogens for the construction of library.The results showed that 73 and 100 sequences were obtained from Nili-Ravi buffalos and Haizi buffalos library,respectively.Based the 97%similarity,sequences of Nili-Ravi buffaloes and Haizi buffaloes were assigned to 20 and 33 OTUs,respectively.For Nili-Ravi buffaloes,70 sequences(17 OTUs)showed≥97%sequence similarity to known species,3 sequences(3 OTUs)had sequence similarity to known species in the range of 93%to 96%.For Haizi buffaloes,70 sequences(15 OTUs)showed≥97%sequence similarity to known species,30 sequences(18 OTUs)had sequence similarity to known species in the range of 84%to 96%.The SGMT and RO clade sequences percentage in Nili-Ravi buffaloes and Haizi buffaloes were 76.71%and 21.91%,76.00%and 19.00%,respectively.Phylogenetic analysis indicated that these sequences were assigned into two large clades.There were 8 OTUs representative sequences from Nili-Ravi buffaloes and 16 OTUs representative sequences from Haizi buffaloes assigned into the same clade on the top of the phylogenetic tree and far from all known species.It could be concluded that Methanobrevibacter genus was the dominant Archaea in the rumen of Nili-Ravi buffaloes and Haizi buffaloes when fed the same diet.The SGMT clade sequences ratio in Nili-Ravi buffaloes almost were the same with Haizi buffaloes,while the RO clade sequences in Haizi buffaloes were lower than that in Nili-Ravi buffaloes,indicating that there were more unknown methanogen sequences in Haizi buffaloes than that in Nili-Ravi buffaloes.