不同日粮饲养黄牛的胃部微生物群落多样性比较分析

为分析不同饲料喂养对黄牛胃中微生物群落结构的影响，分别用芒草单一喂养和农家混合饲料喂养两年的黄牛为实验组和对照组。以瘤胃、蜂巢胃、重瓣胃和皱胃四个胃中的微生物为研究对象，采用原位包埋裂解法和脉冲场电泳（pulsed field gel electrophoresis，PFGE）提取微生物总 DNA，脉冲场电泳（pulsed field gel elec-trophoresis，PFGE）。使用细菌16S rRNA 引物341F ／534R 及真菌18S rDNA 引物 NS1／GCFungi 进行降落 PCR 扩增。变性梯度凝胶电泳（denaturing gradient gel electrophoresis，DGGE）对扩增产物进行区分，使用硝酸银染色。电泳扫描结果通过 Quantity one 软件进行分析，SPSS 软件进行数据统计。针对实验组和对照组瘤胃样品共性条带和特性条带测序并比对。结果表明：实验组与对照组细菌群落结构变化较大，UPGAM聚类图上被分为两支，相似性只有0．35。且香农多样性指数及条带丰度都明显少于对照组。真菌 DGGE 图谱条带差别不大，聚类图上显示实验组四个样品与对照组四个样品相似性在0．43～0．68之间。多样性及条带丰度在实验组与对照组之间差异0．0027～0．5999。测序结果，细菌与数据库中未培养细菌种类较为接近，而真菌中部分与已知菌种接近。芒草单一饲养对牛胃中的微生物群落结构具有极大的影响，对细菌的影响尤为明显。

Comparison of Microbial Community Diversities in S tomach of the Yellow Cattle with Different Feeding Types

As the influence of feeding type on microbial community structure and functions in host is poorly understood, this study was conducted to investigate the variation of the gastric microflora in a variety of yellow cattle fed diets.For 2 years,the experimental group was fed with Miscanthus(Miscanthus sinensis,M.sinensis)only and the control group was fed with total mixed diets.Samples were collected from the rumen,reticulum,omasum,and abomasum.Total microbial DNA was extracted using in situ lysis and pulsed field gel electrophoresis (PFGE).The bacterial 16S rDNA and the fun-gal 18S rDNA were amplified using touchdown PCR,and the denaturing gradient gel electrophoresis (DGGE)profiles were analyzed.Common and unique bands between the experimental and control groups from the rumen were sequenced and compared with GenBank sequences.Results revealed a significant difference in bacterial community structure be-tween the experimental and control groups.An unweighted pair-group method with arithmetic mean (UPGMA)revealed that the 2 groups were clustered in 2 separate branches and had a similarity of only 0.35.Moreover,the Shannon diver-sity index and band abundance were significantly lower in the experimental group than in the control group.The fungal DGGE profile bands showed no significant change.The similarity in the four samples between the experimental and con-trol groups ranged from 0.43 to 0.68.The difference in band abundance and density between the experimental and con-trol groups ranged from 0.0027 to 0.5999.In terms of sequencing results,the bacterial sequences were similar to those of uncultured bacteria in the database,and some fungal sequences were similar to those of known species.In conclu-sion,high cellulose M.sinensis has important influence on microorganisms in the stomach of cattle,which influenced the microbial community structure,especially for the bacterial community.