反刍动物营养 Ruminant Nutrition

基于16SrRNA基因克隆文库技术分析广西富钟水牛瘤胃产甲烷菌组成及多样性

  • 杨承剑 ,
  • 梁辛 ,
  • 韦升菊 ,
  • 李舒露 ,
  • 梁贤威 ,
  • 邹彩霞 ,
  • 杨炳壮 ,
  • 李丽莉
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  • 中国农业科学院广西水牛研究所, 农业部(广西)水牛遗传繁育重点实验室, 南宁 530001

收稿日期: 2014-06-01

  网络出版日期: 2014-12-05

基金资助

广西自然基金青年基金项目(2012GXNSFBA053070);广西水产畜牧兽医局科技计划项目(桂渔牧科1304516);农业部948项目(2011-G26)

Compositon and Diversity of Ruminal Methanogens in Guangxi Fuzhong Buffaloes: an Analysis of Using 16S rRNA Gene Cloning Library Technique

  • YANG Chengjian ,
  • LIANG Xin ,
  • WEI Shengju ,
  • LI Shulu ,
  • LIANG Xianwei ,
  • ZOU Caixia ,
  • YANG Bingzhuang ,
  • LI Lili
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  • Key Laboratory of Buffalo Genetics, Breeding and Reproduction Technology, Ministry of Agriculture (Guangxi), Guangxi Buffalo Research Institute, Chinese Academy of Agricultural Sciences, Nanning 530001, China

Received date: 2014-06-01

  Online published: 2014-12-05

摘要

本研究旨在利用16S rRNA基因克隆库技术分析广西富钟水牛瘤胃产甲烷菌组成及多样性.选取3头体况基本一致的健康雌性富钟水牛作为试验动物,采用机械破壁法提取瘤胃内容物总DNA,采用产甲烷菌引物Met86F/Met1340R扩增16S rRNA基因,构建16S rRNA基因克隆文库.结果表明,本试验共获得93个非嵌合体16S rRNA序列,按照97%的相似性划分为39个分类操作单元(OTU).其中,60个序列(15个OTU)与已培养细菌16S rRNA序列相似性≥97%,占总序列的64.5%;32个序列(23个OTU)与已培养菌16S rRNA序列相似性处于90%~(<97%);仅有1个序列与Methanomassiliicoccus luminyensis相似性<90%.系统发育树分析表明,98.9%的序列均属于甲烷杆菌目(Methanobacteriales),部分序列与Methanobacteriales中任何已知相似序列都相隔较远,它们可能代表Methanobacteriales中新的属或种.由上述结果可见,富钟水牛瘤胃产甲烷菌以Methanobacteriales为优势菌群,其中有许多未知的产甲烷菌需进一步分离培养并对其功能进行分析.

本文引用格式

杨承剑 , 梁辛 , 韦升菊 , 李舒露 , 梁贤威 , 邹彩霞 , 杨炳壮 , 李丽莉 . 基于16SrRNA基因克隆文库技术分析广西富钟水牛瘤胃产甲烷菌组成及多样性[J]. 动物营养学报, 2014 , 26(12) : 3635 -3642 . DOI: 10.3969/j.issn.1006-267x.2014.12.012

Abstract

The objective of this study was to analysis the composition and the diversity of ruminal methanogens in Guangxi Fuzhong buffaloes by using 16S rRNA gene cloning library technique. Three healthy female Fuzhong buffaloes with similar body condition were used in this study. Total DNA of ruminal contents was extracted by bead-beating method. Primers of Met86F/Met1340R of methanogens were used to amplify 16S rRNA gene for the construction of 16S rRNA gene clone library. The results showed that 93 chimera-free 16S rRNA sequences were obtained in the present study. Based the 97% similarity, these sequences were assigned to 39 operational taxonomic units (OTUs). Among those, sixty sequences showed ≥97% sequence similarity to known species (15 OTUs, occupied 64.5% of total sequences), thirty two sequences had sequence similarity to known species in the range of 90% to <97% (23 OTUs), and only one sequence displayed similarity <90% with Methanomassiliicoccus luminyensis. Phylogenetic analysis indicated that 98.9% sequences of methanogens in the rumen of Fuzhong buffaloes belonged to Methanobacteriales. Some sequences were far from all known species on the phylogenetic tree. They might represent new genus or species in Methanobacteriales in the rumen of Fuzhong buffaloes. It can be concluded that Methanobacteriales is the dominant methanogens in the rumen of Fuzhong buffaloes. Further study should be done for the isolation of unknown methanogens and their function analysis.

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