不同年龄麦洼牦牛肠道菌群的分离鉴定及其群落结构的变化

Isolation and identification of gut microbiota with community structure changes in different ages of Maiwa yaks

为了解麦洼牦牛肠道细菌群落结构组成和功能及其动态变化过程,同时获得具有潜在益生菌活性和应用可能性的肠道菌株,采用不同的培养基在不同的培养条件下分离麦洼牦牛肠道细菌并进行16S r DNA分子鉴定,同时采用变性梯度凝胶电泳(denaturing gradient gel electrophoresis,DGGE)技术分析不同年龄麦洼牦牛肠道细菌的群落结构和功能及其差异。结果表明,从不同年龄麦洼牦牛大肠内容物中共分离到90株细菌,其中3株归为放线菌门(Actinobacteria),64株为厚壁菌门(Firmicutes),23株为变形菌门(Proteobacteria);分离到的34株芽孢杆菌属(Bacillus)和14株乳酸杆菌属(Lactobacillus)细菌具有潜在益生菌活性,可作为微生物饲料添加剂补饲麦洼牦牛;对DGGE图谱的UPGMA聚类、PCA和多样性指数分析显示,不同年龄的麦洼牦牛肠道细菌的群落结构有差异,相同年龄的个体相似度较高,而不同年龄的个体相似度较小,较大年龄(2.5和3.5岁)的牦牛肠道菌群多样性大于较小年龄个体(0.5和1.5岁),且分布更均匀,肠道菌群随宿主年龄变化是一个动态变化并趋于稳定的过程;DGGE条带的物种鉴定结果表明,大部分条带为未(能)培养细菌,PCR-DGGE与分离培养法结合可以很好地表征麦洼牦牛肠道菌群的结构和功能。

To understand the community structure composition, functions and dynamic changes of Maiwa yaks＇ gut microbiota and obtain the gut bacterial strain of Maiwa yaks with Potential probiotic activity and application possibility, different culture mediums were used to separate the intestinal bacteria of Maiwa yaks under different culture conditions, and 16S rDNA molecular identification was also carried out. In the meantime, denaturing gradient gel electrophoresis（DGGE） technology was also applied to analyze the community structure and function and the difference of intestinal bacteria of Maiwa yaks at different ages. The results showed that totally 90 strains of bacteria were separated from the gut content of Maiwa yaks at different ages, in which 3, 64, and 23 strains were classified as respectively. Totally 34 strains the phylum of Actinobacteria, Firmicutes and of the genus Bacillus and 14 strains of the genus Proteobacteria Lactobaeillus bacteria with potential probiotic activity were also separated, and they can be used as microbial feed additives to Maiwa yak supplementary feeding. Analysis of UPGMA clustering, PCA, and diversity index of DGGE spectrum indicated that the community structure of gut microbiota at different ages is different, and the similarity between animals of the same ages is high, while the similarity between different ages of animals is low. The diversity of gut microbiota of elder groups of Maiwa yaks （2.5 and 3.5 years） is significantly higher than that of younger groups （0.5 and 1.5 years） but with more homogeneous distribution, which indicates that the change of gut microbiota along with the age change of host is a dynamic process tending to be stable. Species identification results of DGGE bands showed that most of the bands were uncultured bacteria, further indicating that the method of combining PCR-DGGE with plate-culture method can be a more comprehensive way to characterize the structure and function of gut microbiota of Maiwa yaks