采用PCR-DGGE技术分析蒙古羊瘤胃液相和固相细菌的多样性
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摘要
为探讨蒙古羊瘤胃液相和固相细菌的多样性,采用变性梯度凝胶电泳技术结合聚合酶链式反应(PCRDGGE)及条带的克隆测序,比较瘤胃液相和固相细菌的差异,同时采用聚类分析和主成分分析(PCA)方法分析瘤胃液相和固相细菌的多样性。结果表明:1)瘤胃液相和固相样品均含有物种丰富的细菌菌群,但液相比固相具有更高的平均条带数,分别为30条和24条;2)瘤胃液相样品比固相样品具有更高的多样性指数、均匀度和丰富度,其数值分别为3.45、0.90和30.20及3.23、0.84和24.62;3)同一动物个体瘤胃液相样品和固相样品聚类在一起,相似性系数均高达0.81;4)共性条带测序结果表明瘤胃的优势细菌主要是Uncultured rumen bacterium和Uncultured Bacteroidetes bacterium,而特异性条带主要是Alcaligenes sp.。不同动物个体的瘤胃液相和固相均含有丰富的细菌菌群,且物种丰富度均较高;同一动物个体的液相与固相相比具有更高的细菌多样性。
This experiment is to explore the diversity of bacteria associated with liquid phase samples and solid phase samples of rumen contents of Mongolian Sheep.This study choice the research methods of polymerase chain reactiondenaturing gradient gel electrophoresis(PCR-DGGE).The results were analyzed by cluster analysis and PCA.Some common and special bands were identified at the same time.The results showed that both the bacteria from the liquid phase samples and solid phase samples had a high diversity,and the liquid phase samples had higher number of bands compared with the solid phase,were 30 and 24,respectively.Compared with the solid phase samples,the shannon diversity index,evenness and richness of liquid phase samples were higher,were 3.45,0.90,30.20 and 3.23,0.84,24.62,respectively.Samples of rumen liquid phase samples and solid phase samples from the same individual clustered together,and the similarity coefficient were up to 0.81.Uncultured rumen bacterium and Uncultured Bacteroidetes bacterium were predominadt in the rumen of sheep.The special bancterial in rumen content was Alcaligenes sp..Furthermore,both the liquid phase samples and solid phase samples of rumen contents of Mongolian Sheep had abundant bacteria and highest diversity in the liquid phase samples.
This experiment is to explore the diversity of bacteria associated with liquid phase samples and solid phase samples of rumen contents of Mongolian Sheep.This study choice the research methods of polymerase chain reactiondenaturing gradient gel electrophoresis(PCR-DGGE).The results were analyzed by cluster analysis and PCA.Some common and special bands were identified at the same time.The results showed that both the bacteria from the liquid phase samples and solid phase samples had a high diversity,and the liquid phase samples had higher number of bands compared with the solid phase,were 30 and 24,respectively.Compared with the solid phase samples,the shannon diversity index,evenness and richness of liquid phase samples were higher,were 3.45,0.90,30.20 and 3.23,0.84,24.62,respectively.Samples of rumen liquid phase samples and solid phase samples from the same individual clustered together,and the similarity coefficient were up to 0.81.Uncultured rumen bacterium and Uncultured Bacteroidetes bacterium were predominadt in the rumen of sheep.The special bancterial in rumen content was Alcaligenes sp..Furthermore,both the liquid phase samples and solid phase samples of rumen contents of Mongolian Sheep had abundant bacteria and highest diversity in the liquid phase samples.
引文
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[11]倪学勤,曾东,周小秋.采用PCR-DGGE技术分析蛋鸡肠道细菌种群结构及多样性[J].畜牧兽医学报,2008,39(7):955-961
[12]荆元强,宋恩亮,杨维仁,等.牛瘤胃微生物定量测定方法研究进展[J].动物医学进展,2011,32(7):93-96
[13]Cunha I S,Barreto C C,Costa O Y A,et al.Bacteria and archaea community structure in the rumen microbiome of goats(Capra hircus)from the semiarid region of Brazil[J].Anaerobe,2011,17(3):118-124
[14]Fouts D E,Szpakowski S,Purushe J,et al.Next generation sequencing to define prokaryotic and fungal diversity in the bovine rumen[J].Plos One,2012,7(11):e48289
[15]Forsberg C W,Cheng K J,White B A.Polysaccharide degradation in the rumen and large intestine[C]//Mackie R I,White B A.Gastrointestinal Microbiology,New York:Chapman and Hall,1997:319-379
[16]Russell J B,Rychlik J L.Factors that alter rumen microbial ecology[J].Science,2001,292(5519):1119-1122
[17]Carberry C A,Kenny D A,Han S,et al.Effect of phenotypic residual feed intake and dietary forage content on the rumen microbial community of beef cattle[J].Applied and Environmental Microbiology,2012,78(14):4949-4958
[18]Jami E,Mizrahi I.Composition and similarity of bovine rumen microbiota across individual animals[J].Plos One,2012,7(3):e33306
[19]Singh K M,Ahir V B,Tripathi A K,et al.Metagenomic analysis of Surti buffalo(Bubalus bubalis)rumen:A preliminary study[J].Molecular Biology Reports,2012,39(4):4841-4848
[20]孔庆亮,王振勇,柴同杰,等.奶牛瘤胃需氧及兼性厌氧菌的PCR-16SrDNA鉴定及日粮的影响[J].畜牧兽医学报,2008,39(10):1367-1372
[21]曾燕,孙朋,倪学勤,等.采用PCR-DGGE技术分析瘤胃菌群在不同纤维素富集条件下的多样性[J].动物营养学报,2013,25(9):2136-2142
[2]祁茹,林英庭,程明,等.瘤胃微生物区系及其相互关系的研究进展[J].饲料博览,2011(8):9-13
[3]曾燕,孙朋,倪学勤,等.不同纤维素培养基及温度对体外培养瘤胃细菌多样性的影响[J].中国农业大学学报,2013,18(6):148-152
[4]Kittelmann S,Seedorf H,Walters W A,et al.Simultaneous amplicon sequencing to explore co-occurrence patterns of bacterial,archaeal and eukaryotic microorganisms in rumen microbial communities[J].Plos One,2013,8(2):e47879
[5]Martn-Orúe S M,Balcells J,Zakraoui F,et al.Quantification and chemical composition of mixed bacteria harvested from solid fractions of rumen digesta:Effect of detachment procedure[J].Animal Feed Science and Technology,1998,71(3):269-282
[6]Volden H,Mydland L T,Harstad O M.Chemical composition of protozoal and bacterial fractions isolated from ruminal contents of dairy cows fed diets differing in nitrogen supplementation[J].Acta Agriculturae Scandinavica,Section A-Animal Science,1999,49(4):235-244
[7]Edwards J E,Mcewan N R,Travis A J,et al.16Sr DNA library-based analysis of ruminal bacterial diversity[J].Antoie Van Leeuwenhoek,2004,86(3):263-281
[8]Muyzer G,De Waal E C,Uitterlinden A G.Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16SrRNA[J].Applied and Enviromental Microbiology,1993,59(3):695-700
[9]Zhou J,Bruns M A,Tiemdje J M.DNA recovery from soils of diverse composition[J].Appl Environ Microbiol,1996,62(2):316-322
[10]Walter J,Hertel C,Tannock G W,et al.Detection of Lactobacillus,Pediococcus,Leuconostoc and Weissella species in human feces by using group-specific PCR primers and denaturing gradient gel electrophoresis[J].Appl Environ Microb,2001,67:2578-2585
[11]倪学勤,曾东,周小秋.采用PCR-DGGE技术分析蛋鸡肠道细菌种群结构及多样性[J].畜牧兽医学报,2008,39(7):955-961
[12]荆元强,宋恩亮,杨维仁,等.牛瘤胃微生物定量测定方法研究进展[J].动物医学进展,2011,32(7):93-96
[13]Cunha I S,Barreto C C,Costa O Y A,et al.Bacteria and archaea community structure in the rumen microbiome of goats(Capra hircus)from the semiarid region of Brazil[J].Anaerobe,2011,17(3):118-124
[14]Fouts D E,Szpakowski S,Purushe J,et al.Next generation sequencing to define prokaryotic and fungal diversity in the bovine rumen[J].Plos One,2012,7(11):e48289
[15]Forsberg C W,Cheng K J,White B A.Polysaccharide degradation in the rumen and large intestine[C]//Mackie R I,White B A.Gastrointestinal Microbiology,New York:Chapman and Hall,1997:319-379
[16]Russell J B,Rychlik J L.Factors that alter rumen microbial ecology[J].Science,2001,292(5519):1119-1122
[17]Carberry C A,Kenny D A,Han S,et al.Effect of phenotypic residual feed intake and dietary forage content on the rumen microbial community of beef cattle[J].Applied and Environmental Microbiology,2012,78(14):4949-4958
[18]Jami E,Mizrahi I.Composition and similarity of bovine rumen microbiota across individual animals[J].Plos One,2012,7(3):e33306
[19]Singh K M,Ahir V B,Tripathi A K,et al.Metagenomic analysis of Surti buffalo(Bubalus bubalis)rumen:A preliminary study[J].Molecular Biology Reports,2012,39(4):4841-4848
[20]孔庆亮,王振勇,柴同杰,等.奶牛瘤胃需氧及兼性厌氧菌的PCR-16SrDNA鉴定及日粮的影响[J].畜牧兽医学报,2008,39(10):1367-1372
[21]曾燕,孙朋,倪学勤,等.采用PCR-DGGE技术分析瘤胃菌群在不同纤维素富集条件下的多样性[J].动物营养学报,2013,25(9):2136-2142