基于DGGE和T-RFLP分析采食不同粗饲料梅花鹿瘤胃细菌区系

doi:10.3864/j.issn.0578-1752.2014.04.016

摘要/Abstract

摘要： 【目的】细菌区系在梅花鹿（Cervus nippon）瘤胃发酵中发挥着重要作用，然而有关梅花鹿瘤胃细菌区系的研究鲜有报道。研究梅花鹿瘤胃细菌区系，为梅花鹿瘤胃发酵调控提供分子生物学依据。【方法】选取4头2岁龄的装有永久性瘤胃瘘管的成年雄性梅花鹿为研究对象，分别饲喂以柞树叶（OL组，梅花鹿A和B）和玉米秸秆（CS组，梅花鹿C和D）为主要粗饲料的日粮，持续饲喂30 d。通过瘤胃瘘管取瘤胃内固液混合物，提取瘤胃微生物基因组DNA。扩增瘤胃细菌16S rRNA基因V3区以及16S rRNA基因，分别用于DGGE和T-RFLP分析。DGGE图谱进行聚类分析，并切取优势条带进行克隆测序，鉴定瘤胃内细菌组成。根据T-RFLP图谱结果计算细菌群落的多样性、优势度、均匀度和丰富度，通过Microbial Community AnalysisⅢ（MiCAⅢ）数据库推测T-RFs可能代表的微生物种类，并进行T-RFLP图谱的聚类分析。【结果】DGGE图谱聚类表明，CS组和OL组瘤胃细菌区系相似性低于65%，表明粗饲料种类影响梅花鹿瘤胃细菌区系。OL组梅花鹿A和B的DGGE图谱相似度大于70%，CS组梅花鹿C和D的DGGE图谱相似性大于75%，而且同组不同个体之间瘤胃细菌区系存在差异。OL组和CS组分别获得20和24个DGGE特异性条带。序列分析表明，CS组条带可归类为拟杆菌门、厚壁菌门和变形菌门，而OL组条带可归类为拟杆菌门、厚壁菌门、变形菌门和互养菌门。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，梅花鹿D（CS组）具有最高的丰富度、多样性、均匀度和最低的优势度，梅花鹿A和B的各项指数相近但低于梅花鹿D，说明OL组中的粗饲料（柞树叶）影响瘤胃中微生物的相对生物量。梅花鹿C和D的各项指数相差较大而且梅花鹿C的指数低于梅花鹿A和B，表明同组不同个体之间存在差异。81、214、272和308 bp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284 bp的T-RFs为共同条带。根据MiCAⅢ结果，这些T-RFs代表细菌归类于拟杆菌门、厚壁菌门、变形菌门和酸杆菌门。T-RFs图谱聚类表明，4头梅花鹿T-RFs聚为两类，粗饲料来源影响梅花鹿瘤胃细菌T-RFs图谱特征，其中梅花鹿A、B和C的T-RFs特征条带图谱相似。【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。

关键词: 梅花鹿 , 细菌区系 , 普雷沃氏菌 , 单宁

Abstract: 【Objective】Bacterial communities play critical roles in the rumen fermentation of Sika deer (Cervus nippon), while the bacterial composition in the rumen of Sika deer is rarely reported. The objective of present study is to investigate the bacterial diversity in the rumen of sika deer, which can provide a molecular basis for manipulation of rumen fermentation.【Method】Four two year old male rumen-cannulated Sika deers fed oak leaf (OL group, Sika deer A and B) and corn stover (CS group, Sika deer C and D) based diets were used in the present study. After 30 days of feeding, rumen contents including solid and liquid fractions were sampled, and the microbial genomic DNA was extracted. V3 region of ruminal bacterial 16S rRNA gene and 16S rRNA gene was amplified, which was used in the DGGE and T-RFLP analysis, respectively. The clustering analysis was applied to DGGE results. The dominant bands in DGGE profiles were obtained, and then used to clone sequencing in order to indentify the bacterial communities. The results of T-RFLP were also applied to clustering analysis, and the possible bacterial structure was speculated by Microbial Community AnalysisⅢ (MiCAⅢ) dataset. 【Result】The clustering patterns of DGGE revealed that the similarity of bacterial diversity between CS group and OL group was lower than 65%, indicating that the bacterial diversity was affected by forage source. The similarity between Sika deer A and B in the OL group, and between Sika deer C and D in the CS group was greater than 70% and 75%, respectively. In addition, the differences were found between animals in the same group. A total of 20 and 24 unique DGGE bands were obtained from the OL and CS groups, respectively. Sequences analysis of DGGE showed that the bacteria in the OL group were composed of Bacteroides, Firmicutes and Proteobacteria phyla, while they were composed of Bacteroides, Firmicutes, Proteobacteria and Synergistetes phyla in the CS group. 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 bacteria in two groups includes Clostridium spp. and Eubacterium spp.. The results of T-RFLP showed that the highest the highest richness, diversity and evenness indices, and the lowest dominance index were found in Sika deer D (CS group). The diversity indices in Sika deer A and B were comparative, but lower than that in Sika deer D, suggesting that forage (oak leaf) in the OL group affected the relative biomass of rumen microbiome. There were clear discrepancies in the diversity indices of Sika deer C and D, and the diversity indices in Sika deer C were lower than Sika deer A and B, indicating that the intra-individual variation. T-RFs representing 81, 214, 272 and 308 bp in OL group were dominant, 90, 95, 175, 273 and 274 bp were predominant in CS group, and 161, 259, 264, 266 and 284 bp were presented in all animals. According to the results of MiCAⅢ, the possible bacteria represented by these T-RFs could assign to the phyla Bacteroides, Firmicutes, Proteobacteria and Acidobacteria. The clustering analysis of T-RFs showed that two clusters were generated from the T-RFs in the two groups, and the T-RFs profiles in Sika deer A, B and C were similar, indicating that forage sources affected the profiles of T-RFs.【Conclusion】These results suggested that Prevotella spp. were the dominant bacteria in rumen of sika deer. The forage source affected the rumen bacterial communities.

Key words: Sika deer , bacterial structure , Prevotella spp. , tannins

李志鹏1, 姜娜2, 刘晗璐1, 崔学哲1, 荆祎1, 杨福合1, 李光玉1. 基于DGGE和T-RFLP分析采食不同粗饲料梅花鹿瘤胃细菌区系[J]. 中国农业科学, 2014, 47(4): 759-768.

LI Zhi-Peng-1, JIANG Na-2, LIU Han-Lu-1, CUI Xue-Zhe-1, JING Yi-1, YANG Fu-He-1, LI Guang-Yu-1. Analysis of Bacterial Diversity in Rumen of Sika Deer (Cervus nippon) fed Different Forages Using DGGE and T-RLFP[J]. Scientia Agricultura Sinica, 2014, 47(4): 759-768.

0
/ / 推荐

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2014.04.016

https://www.chinaagrisci.com/CN/Y2014/V47/I4/759

参考文献

[1]Wright A D G, Klieve A V. Does the complexity of the rumen microbial ecology preclude methane mitigation? Animal Feed Science and Technology, 2011, 166/167: 248-253.

[2]An D D, Dong X Z, Dong Z Y. Prokaryote diversity in the rumen of yak (Bos grunniens) and Jinnan cattle (Bos taurus) estimated by 16S rDNA homology analyses. Anaerobe, 2005, 11(4): 207-215.

[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. 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. Molecular Biology Reports, 2012, 39(2): 1181-1192.

[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). 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 A D. Molecular diversity of the rumen microbiome of Norwegian Reindeer on natural summer pasture. Microbial Ecology, 2009, 57(2): 335-348.

[7]Sundset M A, Praesteng K E, Cann I K, Mathiesen S D, Mackie R I. Novel rumen bacterial diversity in two geographically separated sub-species of reindeer. Microbial Ecology, 2007, 54(3): 424-438.

[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. 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. Applied and Environmental Microbiology, 2010, 76(22): 7482-7490.

[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. 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. Molecular Biology Reports, 2010, 37(4): 2063-2073.

[13]刘开朗,卜登攀, 王加启, 于萍, 李旦, 赵圣国, 贺云霞, 魏宏阳, 周凌云. 六个不同品种牛的瘤胃微生物群落的比较分析. 中国农业大学学报, 2009, 14(1): 13-18.

Liu K L, 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. Journal of China Agricultural University, 2009, 14(1): 2063-2073. (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. PLoS ONE, 2012, 7(6): e38571.

[15]Goel G, Puniya A K, Aguilar CN, Singh K. Interaction of gut microflora with tannins in feeds. Naturwissenschaften, 2005, 92(11): 497-503.

[16]McSweeney C S, Palmer B, McNeill D M, Krause D O. Microbial interactions with tannins: nutritional consequences for ruminants. 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. Animal Feed Science and Technology, 2005, 121(1/2): 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. Journal of Bacteriology, 1996, 178(19): 5636-5643.

[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. 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. 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). 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. 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 and 18S rRNA genes. 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. Applied and Environmental Microbiology, 2004, 70(8): 4800-4806.

[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. Journal of Microbiological Methods, 2007, 70(3): 565-569.

[25]Watanabe K, Kodama Y, Harayama S. Design and evaluation of PCR primers to amplify bacterial 16S ribosomal DNA fragments used for community fingerprinting. 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. 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 rRNA gene-based analysis. 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. 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 rRNA genes. Metagenomics, 2012, 1: 1-11.

[30]Kim M, Morrison M, Yu Z. Status of the phylogenetic diversity census of ruminal microbiomes. FEMS Microbiology Ecology, 2011, 76(1): 49-63.

[31]Krause D O, 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. 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. Current Microbiology, 2000, 41(1): 45-49.

[33]Kobayashi Y. Inclusion of novel bacteria in rumen microbiology: need for basic and applied science. Animal Science Journal, 2006, 77(4): 375-385.

[34]Orpin C G M S. Microbiology of digestion in the Svalbard reindeer (Rangifer tarandus platyrhynchus). Rangifer, 1990(3): 187-199.

[35]Aagnes T H, Sormo W, Mathiesen S D. Ruminal microbial digestion in free-living, in captive lichen-ded, and in Starved Reindeer(Rangifer Tarandus Tarandus) in winter. Applied and Environmental Microbiology, 1995, 61(2): 583-591.

[36]Tajima K, Aminov R I, Nagamine T, Matsui H, Nakamura M, Benno Y. Diet-dependent shifts in the bacterial population of the rumen revealed with real-time PCR. Applied and Environmental Microbiology, 2001, 67(6): 2766-2774.

[37]Sleat R, Mah R A. Clostridium populeti sp. nov, a cellulolytic species from a wood-biomass digestor. International Journal of Systematic Bacteriology, 1985, 35(2): 160-163.

[38]Taguchi H, Senoura T, Hamada S, Matsui H, Kobayashi Y, Watanabe J, Wasaki J, Ito S. Cloning and sequencing of the gene for cellobiose 2-epimerase from a ruminal strain of Eubacterium cellulosolvens. FEMS Microbiology Letters, 2008, 287(1): 34-40.

[39]Yoda K, Toyoda A, Mukoyama Y, Nakamura Y, Minato H. Cloning, sequencing, and expression of a Eubacterium cellulosolvens 5 gene encoding an endoglucanase (Cel5A) with novel carbohydrate-binding modules, and properties of Cel5A. Applied and Environmental Microbiology, 2005, 71(10): 5787-5793.

[40]Osborne J M, Dehority B A. Synergism in degradation and utilization of intact forage cellulose, hemicellulose, and pectin by three pure cultures of ruminal bacteria. Applied and Environmental Microbiology, 1989, 55(9): 2247-2250.

[41]Min B R, Attwood G T, Reilly K, Sun W, Peters J S, Barry T N, McNabb W C. Lotus corniculatus condensed tannins decrease in vivo populations of proteolytic bacteria and affect nitrogen metabolism in the rumen of sheep. Canadian Journal of Microbiology, 2002, 48(10): 911-921.

[42]李成云, 袁英良, 朴光一. 缩合单宁对瘤胃挥发性脂肪酸及微生物生长的影响. 饲料研究, 2010(11): 5-7.

Li C Y, Yuang Y L, Piao G Y. The effects of condensed tannins on the volatile fatty acids and the growth of microorganism in the rumen. Feed Research, 2010(11): 5-7. (in Chinese)

[43]金龙. 紫色达利菊提取缩合单宁对大肠杆菌和瘤胃氮代谢以及瘤胃微生物的影响[D]. 哈尔滨: 东北农业大学, 2011.

Jin L. Effects of condensed tannins from purple prairie clover on fecal shedding of Escherichia coli by beff cattle and on rumen fermentation and rumen bacteria[D]. Harbin: Northeast Agricultural University, 2011. (in Chinese)

[44]Jones G A, McAllister T A, Muir A D, Cheng K J. Effects of sainfoin (Onobrychis viciifolia Scop.) condensed tannins on growth and proteolysis by four strains of ruminal bacteria. Applied and Environmental Microbiology, 1994, 60(4): 1374-1378.

[45]Ramsak A, Peterka M, Tajima K, Martin JC, Wood J, Johnston M E A, Aminov R I, Flint H J, Avgustin G. Unravelling the genetic diversity of ruminal bacteria belonging to the CFB phylum. FEMS Microbiology Ecology, 2000, 33(1): 69-79.