基于DGGE和T-RFLP分析采食不同粗饲料梅花鹿瘤胃细菌区系
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- 英文论文题名：The Analysis of Bacterial Diversity in the Rumen of Sika Deer(Cervus nippon) fed Different Forages Using DGGE and T-RLFP
- 论文作者：李志鹏 ; 姜娜 ; 刘晗璐 ; 崔学哲 ; 荆祎 ; 杨福合 ; 李光玉
- 英文论文作者：Li Zhipeng ; Jiang Na ; Liu Hanlu ; Cui Xuezhe ; Jing Yi ; Yang Fuhe ; Li Guangyu ; Jilin Provincial Key Labororary for Molecular Biology of Special Economic Animals ; Insitute of Special Animal and Plant Sciences of Chinese Academy of Agricultural Sciences ; Laboratory of Biodiversity and Eco-Agriculture ; Agro-Environmental Protection Institute ; Ministry of Agriculture
- 年：2016
- 作者机构：中国农业科学院特产研究所吉林省特种经济动物分子重点实验室;农业部环境保护科研监测所生态农业研究室;
- 论文关键词：梅花鹿 ; 细菌区系 ; 普雷沃氏菌 ; 单宁
- 英文论文关键词：Sika deer ; bacterial structure ; Prevotella spp ; tannins
- 会议召开时间：2016-06-01
- 会议录名称：“十二五” 鹿科学研究进展
- 语种：中文
- 分类号：S825
- 学会代码：ZXMY
- 学会名称：中国畜牧业协会
- 页数：13
- 文件大小：1336k
- 原文格式：O
摘要
【目的】研究梅花鹿(Cervus nippon)瘤胃细菌区系,为梅花鹿瘤胃发酵调控提供分子生物学依据。【方法】采用DGGE与T-RFLP技术比较以玉米秸秆(CS组)和柞树叶(OL组)为主要粗饲料的梅花鹿瘤胃细菌区系变化。【结果】DGGE图谱聚类表明,OL组与CS组细菌区系相似度低于65%,而且同组不同个体之间瘤胃细菌区系存在差异。序列分析表明,OL组与CS组中存在大量Prevotella spp.,但不同组中Prevotella spp.在种水平组成不同,主要纤维降解菌为Clostridium spp.与Eubacterium spp.T-RFLP结果显示,OL组与CS组以及同组不同梅花鹿瘤胃细菌的丰富度、多样性、均匀度和优势度有差异。81、214、272和308bp的T-RFs为OL组优势条带,90、95、175、273和274 bp的T-RFs为CS组优势条带,161、259、264、266和284bp的T-RFs为共同条带。【结论】Prevotella spp.是梅花鹿瘤胃优势细菌,但不同粗饲料影响梅花鹿瘤胃细菌区系组成。
【Objective】The objective of present study is to investigate the bacterial diversity in the rumen of sika deer(Cervus nippon),which can provide molecular basis for manipulation of rumen fermentation.[Method]The bacterial communities in the rumen of sika deer feeding oak leaves based(OL group) and corn stalk based(CS group) diets were compared using DGGE and T- RFLP.[Result]The clustering patterns of DGGE indicated that the similarity of bacterial diversity between CS group and OL group was lower than 65%,the differences were found between animlas in the same group.Sequneces analysis of DGGE showed that Prevotella spp.were the dominant bacteria in the OL and CS groups,but the composition of genus Prevotella at species level was different in two groups.The dominant fibrolytic bacterial in two groups includes Clostridium spp.and Eubacterium spp..The results of T- RFLP showed that the richness,diversity,evenness and dominance indices between CS group and OL group,and anmilas in the same group were varied.T- RFs representing 81 bp,214bp,272 bp and 308 bp in OL group were dominant,90 bp,95bp,175 bp,273bp and 274 bp were predominant in CS group,and 161 bp,259 bp,264bp,266 bp and 284 bp were presented in all animals.[Conclusion]These results suggested that Prevotella spp.were the dominant bacteria in the rumen of sika deer.The forage source affected the rumen bacterial communities.
【Objective】The objective of present study is to investigate the bacterial diversity in the rumen of sika deer(Cervus nippon),which can provide molecular basis for manipulation of rumen fermentation.[Method]The bacterial communities in the rumen of sika deer feeding oak leaves based(OL group) and corn stalk based(CS group) diets were compared using DGGE and T- RFLP.[Result]The clustering patterns of DGGE indicated that the similarity of bacterial diversity between CS group and OL group was lower than 65%,the differences were found between animlas in the same group.Sequneces analysis of DGGE showed that Prevotella spp.were the dominant bacteria in the OL and CS groups,but the composition of genus Prevotella at species level was different in two groups.The dominant fibrolytic bacterial in two groups includes Clostridium spp.and Eubacterium spp..The results of T- RFLP showed that the richness,diversity,evenness and dominance indices between CS group and OL group,and anmilas in the same group were varied.T- RFs representing 81 bp,214bp,272 bp and 308 bp in OL group were dominant,90 bp,95bp,175 bp,273bp and 274 bp were predominant in CS group,and 161 bp,259 bp,264bp,266 bp and 284 bp were presented in all animals.[Conclusion]These results suggested that Prevotella spp.were the dominant bacteria in the rumen of sika deer.The forage source affected the rumen bacterial communities.
引文
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[3]Hiura T,Hashidoko Y,Kobayashi Y,Tahara S.Effective degradation of tannic acid by immobilized rumen microbes of a sika deer(Cervus nippon yesoensis)in winter[J].Animal Feed Science and Technology,2010,155(1):1-8.
[4]Leng J,Cheng Y M,Zhang C Y,Zhu R J,Yang S L,Gou X,Deng W D,Mao H M.Molecular diversity of bacteria in Yunnan yellow cattle(Bos taurs)from Nujiang region,China[J].Molecular Biology Reports,2012,39(2):1 181-1192.
[5]Pei C X,Liu Q A,Dong C S,Li H Q,Jiang J B,Gao W J.Diversity and abundance of the bacterial 16S rRNA gene sequences in forestomach of alpacas(Lama pacos)and sheep(Ovis aries)[J].Anaerobe,2010,16(4):426-432.
[6]Sundset M,Edwards J,Cheng Y,Senosiain R,Fraile M,Northwood K,Praesteng KE,Glad T,Mathiesen S,Wright AD.Molecular diversity of the rumen microbiome of Norwegian Reindeer on natural summer pasture[J].Microbial Ecology,2009,57(2):335-348.
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[8]张玲.双峰驼瘤胃细菌多样性及宏基因组文库构建与初步筛选[D].乌鲁木齐:新疆农业大学,2010.Zhang L.Bacterial diversity and the metagenome library construction of the rumen from Bactrian camel(Camelus Bactrianus)[D].Urumqi:Xinjiang Agricultural University,2010.(in Chinese)
[9]de Menezes A B,Lewis E,O'Donovan M,O'Neill B F,Clipson N,Doyle E M.Microbiome analysis of dairy cows fed pasture or total mixed ration diets[J].FEMS Microbiology Ecology,2011,78(2):256-265.
[10]Fernando S C,Purvis H T,Najar F Z,Sukharnikov L O,Krehbiel C R,Nagaraja T G,Roe B A,DeSilva U.Rumen microbial population dynamics during adaptation to a high-grain diet[J].Applied and Environmental Microbiology,2010,76(22):7 482-7 490.
[11]Shi P J,Meng K,Zhou Z G,Wang Y R,Diao Q Y,Yao B.The host species affects the microbial community in the goat rumen[J].Letters in Applied Microbiology,2008,46(1):132—135.
[12]Yang S L,Ma S C,Chen J,Mao H M,He Y D,Xi D M,Yang L Y,He T B,Deng W D.Bacterial diversity in the rumen of Gayals(Bos frontalis),Swamp buffaloes(Bubalus bubalis)and Holstein cow as revealed by cloned 16S rRNA gene sequences[J].Molecular Biology Reports,2010,37(4):2 063-2 073.
[13]刘开朗,卜登攀,王加启,等.六个不同品种牛的瘤胃微生物群落的比较分析[J].中国农业大学学报,2009,14(1):13-18.Liu KL,Bu D P,Wang J Q,Yu P,Li D,Zhao S G,He Y X,Wei H Y,Zhou L Y.Comparative analysis of rumen microbial communities in six species of cattle[J].Journal of China Agricultural University,2009,14(1):2 063-2 073.(in Chinese)
[14]Pope P B,Mackenzie A K,Gregor I,Smith W,Sundset M A,McHardy A C,Morrison M,Eijsink V G.Metagenomics of the Svalbard reindeer rumen microbiome reveals abundance of polysaccharide utilization loci[J].PLoS ONE,2012,7(6):e38571.
[15]Goel G,Puniya A K,Aguilar CN,Singh K.Interaction of gut microflora with tannins in feeds[J].Naturwissenschaften,2005,92(11):497-503.
[16]McSweeney C S,Palmer B,McNeill D M,Krause D 0.Microbial interactions with tannins;nutritional consequences for ruminants[J].Animal Feed Science and Technology,2001,91(1/2):83-93.
[17]Min B R,Attwood G T,McNabb W C,Molan A L,Barry T N.The effect of condensed tannins from Lotus corniculatus on the proteolytic activities and growth of rumen bacteria[J].Animal Feed Science and Technology,2005,121(l/2):45-58.
[18]Nubel U,Engelen B,Felske A,Snaidr J,Wieshuber A,Amann R I,Ludwig W,Backhaus H.Sequence heterogeneities of genes encoding 16S rRNAs in Paenibacillus polymyxa detected by temperature gradient gel electrophoresis[J].Journal of Bacteriology,1996,178(19):5 636-5 643.
[19]朱伟云姚文,毛胜勇.变性梯度凝胶电泳法研究断奶仔猪粪样细菌区系变化[J].微生物学报,2003,43(3):503-508.Zhu W Y,Yao W,Mao S Y.Development of bacterial community in faeces of weaning piglets as revealed by denaturing gradient gel electrophoresis[J].Acta Microbiologica Sinica,2003,43(3):503-508.(in Chinese)
[20]Hongoh Y,Ohkuma M,Kudo T.Molecular analysis of bacterial microbiota in the gut of the termite Reticulitermes speratus(Isoptera;Rhinotermitidae)[J].FEMS Microbiology Ecology,2003,44(2):231-242.
[21]Saeed A I,Sharov V,White J,Li J,Liang W,Bhagabati N,Braisted J,Klapa M,Currier T,Thiagarajan M,Sturn A,Snuffin M,Rezantsev A,Popov D,Ryltsov A,Kostukovich E,Borisovsky I,Liu Z,Vinsavich A,Trush V,Quackenbush J.TM4:a free,open-source system for microarray data management and analysis[J].BioTechniques,2003,34(2):374-378.
[22]Shyu C,Soule T,Bent SJ,Foster J A,Forney L J.MiCA:a web-based tool for the analysis of microbial communities based on terminal-restriction fragment length polymorphisms of 16S and18SrRNA genes[J].Microbial Ecology,2007,53(4):562-570.
[23]Yu Z,Morrison M.Comparisons of different hypervariable regions of rrs genes for use in fingerprinting of microbial communities by PCR-denaturing gradient gel electrophoresis[J].Applied and Environmental Microbiology,2004,70(8):4 800-4 806.
[24]Huws S A,Edwards J E,Kim E J,Scollan N D.Specificity and sensitivity of eubacterial primers utilized for molecular profiling of bacteria within complex microbial ecosystems[J].Journal of Microbiological Methods,2007,70(3):565-569.
[25]Watanabe K,Kodama Y,Harayama S.Design and evaluation of PCR primers to amplify bacterial16S ribosomal DNA fragments used for community fingerprinting[J].Journal of Microbiological Methods,2001,44(3):253-262.
[26]Osborn A M,Moore E R,Timmis K N.An evaluation of terminal-restriction fragment length polymorphism(T-RFLP)analysis for the study of microbial community structure and dynamics[J].Environmental Microbiology,2000,2(1):39-50.
[27]Bekele A Z,Koike S,Kobayashi Y.Genetic diversity and diet specificity of ruminal Prevotella revealed by 16S rRN A gene-based analysis[J].FEMS Microbiology Letters,2010,305(1):49-57.
[28]Stevenson D M,Weimer P J.Dominance of Prevotella and low abundance of classical ruminal bacterial species in the bovine rumen revealed by relative quantification real-time PCR[J].Applied Microbiology and Biotechnology,2007,75(1):165-174.
[29]Wu S,Baldwin R L,Li W,Li C,Connor E E,Li R W.The bacterial community domposition of the bovine rumen detected using pyrosequencing of 16S rRN A genes[J].Metagenomics,2012,1:1-11.
[30]Kim M,Morrison M,Yu Z.Status of the phylogenetic diversity census of ruminal microbiomes[J].FEMS Microbiology Ecology,2011,76(1):49-63.
[31]Krause D 0,Denman S E,Mackie R I,Morrison M,Rae A L,Attwood G T,McSweeney C S.Opportunities to improve fiber degradation in the rumen:microbiology,ecology,and genomics[J].FEMS Microbiology Reviews,2003,27(5):663-693.
[32]Matsui H,Ogata K,Tajima K,Nakamura M,Nagamine T,Aminov RI,Benno Y.Phenotypic characterization of polysaccharidases produced by four Prevotella type strains[J].Current Microbiology,2000,41(1):45-49.
[33]Kobayashi Y.Inclusion of novel bacteria in rumen microbiology:need for basic and applied science[J].Animal Science Journal,2006,77(4):375-385.
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