林麝粪便细菌多样性研究
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摘要
为研究林麝粪便细菌的结构与组成,选取6只2~3岁的圈养条件相同且健康的公林麝,饲喂精粗比为2∶8的饲粮,收集其新鲜粪便,提取其总DNA,然后用16SrRNA细菌通用引物进行PCR扩增,并用Illumina Miseq250PE测序平台,对扩增子进行Miseq双端测序。结果显示:1)借助Illumina Miseq250PE测序平台对样品进行测序,共获得85 091条有效序列,5 133个有效OTU。2)所得OTU经物种注释被归类为23个门,其中厚壁菌门(Firmicutes)(51.1%±19.9%)、拟杆菌门(Bacteroidetes)(35.6%±15.5%)、变形菌门(Proteobacteria)(5.2%±5.6%)和未分类菌门(*Unassigned)(2.2%±1.0%)为优势菌门,其总和达到菌群相对含量的94.1%;将其进一步分为240个菌属,其中18个为共享菌属,拟杆菌目(*Bacteroidales)(16.5%±10.2%)、瘤胃菌科(*Ruminococcaceae)(14.7%±13.8%)和梭菌目(*Clostridiales)(13.7%±3.9%)含量相对较高。3)OTUs稀释曲线均未达到平台期,说明测序深度不足以覆盖所有的菌群。4)非加权聚类图(PUGMA)显示样品B和E、C和F的菌群多样性相似度较高,而样品A和其他样品的相似度相对较低。由此可见,在正常状态下,健康林麝粪便分布有23个门类细菌群落,以Firmicutes、Bacteroidetes和Proteobacteria为优势菌群,进一步将林麝粪便细菌群落细分为240个属且发现林麝个体间菌群差异性较小且与其他草食动物优势菌群结构相似。
To investigate the diversity of the male musk deer hindgut bacterial community,healthy musk deer(n=6;2-3years of age)were fed using the diet consisting of 20% concentrate and 80% roughage ad libitum.Then we sampled flesh feces and extracted total DNA.The universal bacterial primers were used to amplify the V4-V5 region of16SrRNA genes.We identified and characterized the overall fecal bacteria composition by all musk deer using the 16 S rRNA high-throughput sequencing on the Illumina Miseq sequencing platform.Results showed that the sequencing data exhibited that the overall number of OTUs and valid reads using the Miseq sequencing of 16 SrRNA gene reached 5 133OTUs(similarity≥97%)and 85 091 reads,respectively.In total,23 phyla were detected in the samples.Among them,the Firmicutes(51.1% ±19.9%),Bacteroidetes(35.6% ±15.5%),*Unassigned(2.2% ±1.0%)and Proteobacteria(5.2%±5.6%)were the dominant phyla which consisted 94.1% of the overall bacteria community.At the genus level,240 genera could be found in all samples or part of samples.Among genera,the 18 shared genera were present in all fecal samples,and the*Bacteroidales(16.5% ±10.2%)、*Ruminococcaceae(14.7% ±13.8%)、*Clostridiales(13.7% ±3.9%)are the predominant shared genera.The OTU rarefaction curves didn't reach thesaturation plateau in the samples reflecting there were not enough coverage estimations.Furthermore,UPGMA analysis of the samples showed that the similarity of B and E、C and F bacteria communities were the highest.Therefore,our research revealed that hindgut bacteria communities from healthy musk deer fed using the basal diet were classified 23 phyla.Among them,Firmicutes,Bacteroidetes and Proteobacteria were regarded as the dominant hindgut bacteria phyla.Further genus-level analysis showed that 240 genera were discovered across all stool samples,18 genera of which were common bacteria communities.The similarity of hindgut bacteria distribution among different musk deer was relatively high,which was also similar with the dominant hindgut bacteria communities from the other herbivores.
To investigate the diversity of the male musk deer hindgut bacterial community,healthy musk deer(n=6;2-3years of age)were fed using the diet consisting of 20% concentrate and 80% roughage ad libitum.Then we sampled flesh feces and extracted total DNA.The universal bacterial primers were used to amplify the V4-V5 region of16SrRNA genes.We identified and characterized the overall fecal bacteria composition by all musk deer using the 16 S rRNA high-throughput sequencing on the Illumina Miseq sequencing platform.Results showed that the sequencing data exhibited that the overall number of OTUs and valid reads using the Miseq sequencing of 16 SrRNA gene reached 5 133OTUs(similarity≥97%)and 85 091 reads,respectively.In total,23 phyla were detected in the samples.Among them,the Firmicutes(51.1% ±19.9%),Bacteroidetes(35.6% ±15.5%),*Unassigned(2.2% ±1.0%)and Proteobacteria(5.2%±5.6%)were the dominant phyla which consisted 94.1% of the overall bacteria community.At the genus level,240 genera could be found in all samples or part of samples.Among genera,the 18 shared genera were present in all fecal samples,and the*Bacteroidales(16.5% ±10.2%)、*Ruminococcaceae(14.7% ±13.8%)、*Clostridiales(13.7% ±3.9%)are the predominant shared genera.The OTU rarefaction curves didn't reach thesaturation plateau in the samples reflecting there were not enough coverage estimations.Furthermore,UPGMA analysis of the samples showed that the similarity of B and E、C and F bacteria communities were the highest.Therefore,our research revealed that hindgut bacteria communities from healthy musk deer fed using the basal diet were classified 23 phyla.Among them,Firmicutes,Bacteroidetes and Proteobacteria were regarded as the dominant hindgut bacteria phyla.Further genus-level analysis showed that 240 genera were discovered across all stool samples,18 genera of which were common bacteria communities.The similarity of hindgut bacteria distribution among different musk deer was relatively high,which was also similar with the dominant hindgut bacteria communities from the other herbivores.
引文
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[13]Wu S,Wang G,Angert E R,Wang W,Li W,Zou H.Composition,diversity,and origin of the bacterial community in grass carp intestine[J].Plos One,2012,7(2):e30440
[14]Gulino L M,Ouwerkerk D,Kang A Y H,Maguire A J,Kienzle M,Klieve A V.Shedding light on the microbial community of the macropod foregut using 454-Amplicon pyrosequencing[J].Plos One,2013,8(4):e61463
[15]Jami E,Israel A,Kotser A,Mizrahi I.Exploring the bovine rumen bacterial community from birth to adulthood[J].The International Society for Microbial Ecology Journal,2013,7(6):1069-1079
[16]Ley R E,Hamady M,Lozupone C,Turnbaugh P J,Ramey R R,Bircher J S,Schlegel M L,Tucker T A,Schrenzel M D,Knight R,Gordon J I.Evolution of mammals and their gut microbes[J].Science,2008,320(5883):1647-1651
[17]Pope P B,Denman S E,Jones M,Tringe S G,Barry K,Malfatti S A,McHardy A C,Cheng J F,Hugenholtz P,McSweeney C S,Morrison M.Adaptation to herbivory by the Tammar wallaby includes bacterial and glycoside hydrolase profiles different from other herbivores[J].Proceedings of the National Academy of Sciences,2010,107(33):14793-14798
[18]Jami E,Mizrahi I.Composition and similarity of bovine rumen microbiota across individual animals[J].Plos One,2012,7(3):e33306
[19]Koike S,Yoshitani S,Kobayashi Y,Tanaka K.Phylogenetic analysis of fiber-associated rumen bacterial community and PCR detection of uncultured bacteria[J].Federation of European Microbiological Societies Microbiology Letters,2003,229(1):23-30
[20]Larue R,Yu Z,Parisi V A,Egan A R,Morriso M.Novel microbial diversity adherent to plant biomass in the herbivore gastrointestinal tract,as revealed by ribosomal intergenic spacer analysis and rrs gene sequencing[J].Environmental Microbiology,2005,7(4):530-543
[21]李志鹏,刘晗璐,崔学哲,荆祎,鲍坤,徐超,杨福合,李光玉.基于16SrRNA基因序列分析梅花鹿瘤胃细菌多样性[J].动物营养学报,2013,25(9):2044-2050(in Chinese)Li Z P,Liu H L,Cui X Z,Jing W,Bao K,Xu C,Yang F H,Li G Y.Bacterial diversity in rumen of sika deer(cervus nippon)based on 16srRNA sequences analysis[J].Chinese Journal of Animal Nutrition,2013,25(9):2044-2050
[22]Chen Y,Penner G B,Li M,Ob,M.Changes in bacterial diversity associated with epithelial tissue in the beef cow rumen during the transition to a high-grain diet[J].Applied and Environmental Microbiology,2011,77(16):5770-5781
[23]Mackie R I,White B A.Recent advances in rumen microbial ecology and metabolism:potential impact on nutrient output[J].Journal of Dairy Science,1990,73(10):2971-2995
[24]Perumbakkam S,Mitchell E A,Craig A M.Changes to the rumen bacterial population of sheep with the addition of 2,4,6-trinitrotoluene to their diet[J].Antonie Van Leeuwenhoek,2011,99(2):231-240
[2]周瑜,赵克雷,田永强,李旭鑫,张修月,王红宁,岳碧松.几种药物对圈养林麝化脓隐秘杆菌菌膜的影响[J].四川动物,2013(3):005Zhou Y,Zhao K L,Tian Y Q,Li X X,Zhang X Y,Wang H N,Yue B S.Effect of drugs on biofilm production among trueperella pyogenes recovered from abscesses of forest musk deer[J].Sichuan Journal of Zoology,2013(3):005(in Chinese)
[3]许珂,卜书海,梁宗锁,王洪永,罗承军,朱承嗣.林麝研究进展[J].黑龙江畜牧兽医,2014(7):051Xu K,Bu S H,Liang Z S,Wang H Y,Luo C J,Zhu C S.Research progress in forest musk deer[J].Heilongjiang Animal Science and Veterinary Medicine,2014(7):051(in Chinese)
[4]赵克雷,李旭鑫,曾博,张修月,岳碧松.圈养林麝脓肿病病原菌分离鉴定及药敏分析[J].四川动物,2011,30(4):522-526Zhao K L,Li X X,Zeng B,Zhang X Y,Yue B S.Isolation and identification of purulent bacteria from forest musk deer and analysis of drug sensitivity[J].Sichuan Journal of Zoology,2011,30(4):522-526(in Chinese)
[5]刘春燕.林麝(Moschus berezovskii)养殖种群死亡原因及其生命生理特征研究[D].上海:华东师范大学,2008Liu C Y.Study on reasons of death and physiological features for breeding forest musk deer[D].Shanghai:East China Normal University.2008(in Chinese)
[6]马炳存.林麝肠道部分正常微生物的分离,鉴定,分型及微生态制剂的研制[D].雅安:四川农业大学,2012Ma B C.Isolation and identification and type-confirmation of forest musk deer intestinal microflora and research of a novel microbial ecological agent[D].Yaan:Sichuan Agricultural University,2012(in Chinese)
[7]Gruninger R J,Sensen C W,McAllister T A,Forster R J.Diversity of rumen bacteria in Canadian cervids[J].Plos One,2014,9(2):e89682
[8]Pereira L B,Vicentini R,Ottoboni L M.Changes in the bacterial community of soil from a neutral mine drainage channel[J].Plos One,2014,9(5):e96605
[9]Srinivasan S,Hoffman N G,Morgan M T,Matsen F A,Fiedler T L,Hall R W,Ross F J,McCoy C O,Bumgarner R,Marrazzo J M,Fredricks D N.Bacterial communities in women with bacterial vaginosis:High resolution phylogenetic analyses reveal relationships of microbiota to clinical criteria[J].Plos One,2012,7(6):e37818
[10]Fliegerova K,Tapio I,Bonin A,Mrazek J,Callegari M L,Bani P,Bayat A,Vilkki J,Kopeˇcny'J,Shingfield K J,Boyer F,Coissac E,Taberlet P,Wallace R J.Effect of DNA extraction and sample preservation method on rumen bacterial population[J].Anaerobe,2014,29:80-84
[11]Edgar R C.Search and clustering orders of magnitude faster than BLAST[J].Bioinformatics,26(19):2460-2461.
[12]Caporaso J G,Bittinger K,Bushman F D,DeSantis T Z,Andersen G L,Knight R.PyNAST:A flexible tool for aligning sequences to a template alignment[J].Bioinformatics,2010,26(2):266-267
[13]Wu S,Wang G,Angert E R,Wang W,Li W,Zou H.Composition,diversity,and origin of the bacterial community in grass carp intestine[J].Plos One,2012,7(2):e30440
[14]Gulino L M,Ouwerkerk D,Kang A Y H,Maguire A J,Kienzle M,Klieve A V.Shedding light on the microbial community of the macropod foregut using 454-Amplicon pyrosequencing[J].Plos One,2013,8(4):e61463
[15]Jami E,Israel A,Kotser A,Mizrahi I.Exploring the bovine rumen bacterial community from birth to adulthood[J].The International Society for Microbial Ecology Journal,2013,7(6):1069-1079
[16]Ley R E,Hamady M,Lozupone C,Turnbaugh P J,Ramey R R,Bircher J S,Schlegel M L,Tucker T A,Schrenzel M D,Knight R,Gordon J I.Evolution of mammals and their gut microbes[J].Science,2008,320(5883):1647-1651
[17]Pope P B,Denman S E,Jones M,Tringe S G,Barry K,Malfatti S A,McHardy A C,Cheng J F,Hugenholtz P,McSweeney C S,Morrison M.Adaptation to herbivory by the Tammar wallaby includes bacterial and glycoside hydrolase profiles different from other herbivores[J].Proceedings of the National Academy of Sciences,2010,107(33):14793-14798
[18]Jami E,Mizrahi I.Composition and similarity of bovine rumen microbiota across individual animals[J].Plos One,2012,7(3):e33306
[19]Koike S,Yoshitani S,Kobayashi Y,Tanaka K.Phylogenetic analysis of fiber-associated rumen bacterial community and PCR detection of uncultured bacteria[J].Federation of European Microbiological Societies Microbiology Letters,2003,229(1):23-30
[20]Larue R,Yu Z,Parisi V A,Egan A R,Morriso M.Novel microbial diversity adherent to plant biomass in the herbivore gastrointestinal tract,as revealed by ribosomal intergenic spacer analysis and rrs gene sequencing[J].Environmental Microbiology,2005,7(4):530-543
[21]李志鹏,刘晗璐,崔学哲,荆祎,鲍坤,徐超,杨福合,李光玉.基于16SrRNA基因序列分析梅花鹿瘤胃细菌多样性[J].动物营养学报,2013,25(9):2044-2050(in Chinese)Li Z P,Liu H L,Cui X Z,Jing W,Bao K,Xu C,Yang F H,Li G Y.Bacterial diversity in rumen of sika deer(cervus nippon)based on 16srRNA sequences analysis[J].Chinese Journal of Animal Nutrition,2013,25(9):2044-2050
[22]Chen Y,Penner G B,Li M,Ob,M.Changes in bacterial diversity associated with epithelial tissue in the beef cow rumen during the transition to a high-grain diet[J].Applied and Environmental Microbiology,2011,77(16):5770-5781
[23]Mackie R I,White B A.Recent advances in rumen microbial ecology and metabolism:potential impact on nutrient output[J].Journal of Dairy Science,1990,73(10):2971-2995
[24]Perumbakkam S,Mitchell E A,Craig A M.Changes to the rumen bacterial population of sheep with the addition of 2,4,6-trinitrotoluene to their diet[J].Antonie Van Leeuwenhoek,2011,99(2):231-240