圈养老年大熊猫肠道菌群16S rDNA克隆文库的建立
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摘要
为了研究老年大熊猫肠道菌群的构成,本试验对3只圈养老年大熊猫粪便细菌构建16SrDNA克隆文库,采用限制性内切酶HinfⅠ、MspⅠ对其进行限制性片段长度多态性(restriction fragment length polymorphism,RFLP)及测序分析。结果显示,老年大熊猫肠道细菌主要由变形菌门和厚壁菌门组成;变形菌门中又以大肠埃希氏菌属为主,其次为假单胞菌属、志贺氏菌属、气单胞菌属;而厚壁菌门中以链球菌属为主,其次为魏斯氏菌属、梭菌属;此外还发现一定比例的未培养细菌。本试验第一次建立了较丰富的老年大熊猫肠道菌群的克隆文库,为分析比较各年龄层大熊猫的肠道菌群结构的异同提供了参照,也为合理饲喂老年大熊猫,保障老年大熊猫的健康提供重要信息。
In order to comparatively analyze the differences and similarities of intestinal flora from all ages of giant pandas,we built 16 SrDNA clone library of three captive elderly giant pandas' fecal bacteria and made restriction fragment length polymorphism(RFLP)and sequencing analysis by using restriction enzymes HinfⅠ and MspⅠ.The results showed that the intestinal flora of elderly giant panda were mainly attributable to two bacteria:Proteobacteria and Firmicutes.Between them,the main in Proteobacteria of intestinal flora of elderly giant panda were Escherichia coli,followed by Pseudomonas sp.,Shigellaand Aeromonas;The main in Firmicutes were Strep-tococcus,followed by Weissella,Clostridium;Of which there were a certain proportion of bacteria were uncultured bacteria.The article firstly established a rich clone library of intestinal flora from elderly giant pandas,which provided a reference to comparatively analyze the similarities and differences in structure of intestinal flora from all ages of giant pandas,as well as provided the important information about reasonable feeding elderly giant pandas and protecting health of elderly giant pandas.
In order to comparatively analyze the differences and similarities of intestinal flora from all ages of giant pandas,we built 16 SrDNA clone library of three captive elderly giant pandas' fecal bacteria and made restriction fragment length polymorphism(RFLP)and sequencing analysis by using restriction enzymes HinfⅠ and MspⅠ.The results showed that the intestinal flora of elderly giant panda were mainly attributable to two bacteria:Proteobacteria and Firmicutes.Between them,the main in Proteobacteria of intestinal flora of elderly giant panda were Escherichia coli,followed by Pseudomonas sp.,Shigellaand Aeromonas;The main in Firmicutes were Strep-tococcus,followed by Weissella,Clostridium;Of which there were a certain proportion of bacteria were uncultured bacteria.The article firstly established a rich clone library of intestinal flora from elderly giant pandas,which provided a reference to comparatively analyze the similarities and differences in structure of intestinal flora from all ages of giant pandas,as well as provided the important information about reasonable feeding elderly giant pandas and protecting health of elderly giant pandas.
引文
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[21]王燚,何廷美,彭广能,等.不同季节亚成体大熊猫肠道菌群ERIC-PCR指纹图谱分析[J].中国兽医科学,2011,41(8):778-783.
[22]熊焰,李德生,王印,等.卧龙自然保护区大熊猫粪样菌群的分离鉴定与分布研究[J].畜牧兽医学报,2000,31(2):165-17l.
[2]邹兴淮,曾鲁军,孙中武,等.大熊猫疾病死亡因素分析及其保护对策[J].东北林业大学学报,1998,26(1):53-56.
[3]黄菁华,张荣昌,申玉军.16SrDNA基因检测技术在肠道微生态研究中的应用[J].山东畜牧兽医,2007,29(2):49-51.
[4]于超,郭海勇,魏嘉良,等.16S~23SrRNA基因序列在细菌鉴定中的应用[J].中国畜牧兽医,2012,39(2):57-59.
[5]王晓丹,李艳红.分子生物学方法在水体微生物生态研究中的应用[J].微生物学通报,2007,34(4):777-781.
[6]崔婷婷,何廷美,彭广能,等.成年大熊猫肠道菌群多样性的16SrDNA-RFLP技术研究[J].中国兽医科学,2012,42(11):991-997.
[7]Reysenbach L,Giver L,Wickham G.Differential amplification of rRNA genes by polymerase chain reaction[J].Appl Environ Microbiol,1992,58(10):3417-3418.
[8]刘玮琦,茆振川,杨宇,等.应用16SrDNA基因文库技术分析土壤细菌群落的多样性[J].微生物学报,2008,48(10):1344-1350.
[9]Good I L.The population frequencies of species and the estimation of population parameters[J].Biometrika,1953,40(3):237-264.
[10]Zhang W,Ki J S,Qian P Y.Microbial diversity in polluted harbor sedimentsⅠ:Bacterial community assessment based on four clone libraries of 16SrDNA[J].Estuar,Coast Shelf Sci,2008,76(3):668-681.
[11]乌兰,葛刚,龚世杰,等.潘阳湖老爷庙水域细菌群落组成分析[J].长江流域资源与环境,2011,20(8):964-969.
[12]张文平,薛正晟,张志和,等.大熊猫肠道菌群结构研究[A].四川省动物学会第九次会员代表大会暨第十届学术研讨会论文集[C].2011.
[13]Apajalahti J H,Srkilahti L K,Mki B R,et al.Effective recovery of bacterial DNA and percent-guanine-puls-cytosine-based analysis of community structure in the gastrointestinal tract of broiler chickens[J].Appl Environ Microbiol,1998,64(10):4084-4088.
[14]倪学勤,Gong J,Yu H,等.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.
[15]鲁海峰,魏桂芳,李仲逵,等.ERIC-PCR分子杂交技术分析大熊猫肠道菌群结构[J].中国微生态学杂志,2005,17(2):81-84.
[16]Wei G F,Lu H F,Zhou Z H,et al.The microbial community in the feces of the giant panda(Ailuropoda melanoleuca)as determined by PCR-TGGE profiling and clone library analysis[J].Microbial Ecology,2007,54(1):194-202.
[17]凌代文.乳酸菌分类鉴定及实验方法[M].北京:中国轻工业出版社,1999.
[18]杨起恒.乳酸菌发酵机理及酸奶工艺优化研究[D].北京:中国农业大学,2005.
[19]李中华,段雄波.魏斯菌属细菌的分类与鉴定新进展[J].国际检验医学杂志,2006,27(8):721-723.
[20]Bjorkroth K J,Schillinger U,Geisen R,et al.Taxonomic study of Weissella confuse and description of Weissella cibaria sp.nov.,detected in food and clinical samples[J].Int J Syst Evol Microbiol,2002,52(1):141-148.
[21]王燚,何廷美,彭广能,等.不同季节亚成体大熊猫肠道菌群ERIC-PCR指纹图谱分析[J].中国兽医科学,2011,41(8):778-783.
[22]熊焰,李德生,王印,等.卧龙自然保护区大熊猫粪样菌群的分离鉴定与分布研究[J].畜牧兽医学报,2000,31(2):165-17l.