大熊猫肠道放线菌的种群组成及多样性分析
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摘要
【目的】探究不同年龄、不同性别大熊猫肠道放线菌的多样性及群落结构,为寻找潜在产生活性化合物的放线菌资源提供理论依据。【方法】采用PCR-DGGE技术对大熊猫肠道放线菌进行分析,对电泳结果进行UPGMA聚类分析、主成分分析、生物多样性等多重比较。【结果】变性梯度凝胶电泳(DGGE)图谱显示,不同大熊猫肠道中放线菌的多样性及群落结构存在明显差异。随着年龄的增长,雌性大熊猫肠道中放线菌的多样性指数(H')和丰富度(S)逐渐减少,而雄性大熊猫肠道内放线菌的多样性指数(H')和丰富度(S)逐渐增多。不同个体的大熊猫肠道放线菌的群落结构存在明显差异,但相同性别之间的相似性很高。DGGE条带回收测序结果表明,获得的28条序列归属于10个放线菌属,其中链霉菌属(Streptomyces)为优势菌属,占总数的46%;北里孢菌属(Kitasatospora)、红球菌属(Rhodococcus)、棒杆菌属(Corynebacterium)、迪茨氏菌属(Dietziaceae)、大理石雕菌属(Marmoricola)、布登堡菌属(Beutenbergia)、微杆菌属(Microbacterium)、链嗜酸菌属(Streptacidiphilus)和芽生球菌属(Blastococcus)等为非链霉菌属,占总数的54%。【结论】大熊猫肠道内蕴藏着丰富的放线菌资源,其肠道菌群的结构与组成受年龄和性别的影响。
[Objective] Study the diversity and community structure of gut actinobacteria of giant panda with the different age and gender, in order to provide scientific basis for searching the actinobacteria possessing potential to produce bioactive compounds. [Methods] Denaturing gradient gel electrophoresis(DGGE) was employed to reveal the gut actinobacteria diversity and community structure of giant panda, and the results were analyzed by multiple comparisons, including UPGMA cluster, PCA and biological diversity of gut actinobacteria in different age and gender. [Results] The diversity and community structure of gut actinobacteria in different panda exhibited significant differences by DGGE analysis. The Shannon-Wiener index(H') and Richness(S) of gut actinobacteria in female giant pandas decreased as they grew older, but the male giant pandas were opposite. The different diversity of gut actinobacteria were observed in different giant panda. However, the diversity index of gut actinobacteria from the same gender possessed higher similarity. The recovery of DGGE strip series showed the 16 S r RNA gene sequence similarity between the 28 stronger bands and the typical strains published in Gen Bank was 96% to 100%, and those sequences belonged to ten genera, Streptomyces was dominant genera accounting for 46% of all sequences, the remainder belonged to genera Kitasatospora, Rhodococcus, Corynebacterium, Dietziaceae, Marmoricola, Beutenbergia, Microbacterium, Streptacidiphilus, Blastococcus, which account for 54%. [Conclusion] This result revealed the intestinal actinobacteria community structure and diversity of giant panda were different and abundant, which were influenced by their age and gender.
[Objective] Study the diversity and community structure of gut actinobacteria of giant panda with the different age and gender, in order to provide scientific basis for searching the actinobacteria possessing potential to produce bioactive compounds. [Methods] Denaturing gradient gel electrophoresis(DGGE) was employed to reveal the gut actinobacteria diversity and community structure of giant panda, and the results were analyzed by multiple comparisons, including UPGMA cluster, PCA and biological diversity of gut actinobacteria in different age and gender. [Results] The diversity and community structure of gut actinobacteria in different panda exhibited significant differences by DGGE analysis. The Shannon-Wiener index(H') and Richness(S) of gut actinobacteria in female giant pandas decreased as they grew older, but the male giant pandas were opposite. The different diversity of gut actinobacteria were observed in different giant panda. However, the diversity index of gut actinobacteria from the same gender possessed higher similarity. The recovery of DGGE strip series showed the 16 S r RNA gene sequence similarity between the 28 stronger bands and the typical strains published in Gen Bank was 96% to 100%, and those sequences belonged to ten genera, Streptomyces was dominant genera accounting for 46% of all sequences, the remainder belonged to genera Kitasatospora, Rhodococcus, Corynebacterium, Dietziaceae, Marmoricola, Beutenbergia, Microbacterium, Streptacidiphilus, Blastococcus, which account for 54%. [Conclusion] This result revealed the intestinal actinobacteria community structure and diversity of giant panda were different and abundant, which were influenced by their age and gender.
引文
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[2]Ley RE,Hamady M,Lozupone C,et al.Evolution of mammals and their gut microbes[J].Science,2008,320(5883):1647-1651
[3]Jin HZ,Li KB.Progress in the study of human intestinal microecosystem[J].Nature Magazine,2004,26(2):88-91(in Chinese)金红芝,李堃宝.人肠道微生态系统的研究进展[J].自然杂志,2004,26(2):88-91
[4]Gandhimathi R,Arunkumar M,Selvin J,et al.Antimicrobial potential of sponge associated marine actinomycetes[J].Journal de Mycologie Médicale/Journal of Medical Mycology,2008,18(1):16-22
[5]Matsui T,Tanaka J,Namihira T,et al.Antibiotics production by an actinomycete isolated from the termite gut[J].Journal of Basic Microbiology,2012,52(6):731-735
[6]Cao YR.Study on the diversity and biological activity of fecal actinobacteria[D].Yangling:Doctoral Dissertation of Northwest Agriculture and Forestry University,2012(in Chinese)曹艳茹.动物粪便放线菌多样性及生物活性研究[D].杨凌:西北农林科技大学博士学位论文,2012
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[8]Zhan XJ,Li M,Zhang ZJ,et al.Molecular censusing doubles giant panda population estimate in a key nature reserve[J].Current Biology,2006,16(12):R451-R452
[9]Xue ZS,Zhang WP,Wang LH,et al.The bamboo-eating giant panda harbors a carnivore-like gut microbiota,with excessive seasonal variations[J].MBio,2015,6(3):e00022-15
[10]Bao N,Liu CJ,Tan Z,et al.Changes of intestinal microflora in the giant panda returned to wild[J].Animal Husbandry&Veterinary Medicine,2005,37(8):10-13(in Chinese)鲍楠,刘成君,谭志,等.野外放归大熊猫肠道菌群变化的研究[J].畜牧与兽医,2005,37(8):10-13
[11]Zhu LF,Wu Q,Dai JY,et al.Evidence of cellulose metabolism by the giant panda gut microbiome[J].Proceedings of the National Academy of Sciences of the United States of America,2011,108(43):17714-17719
[12]Zhou JL,Li FP,Liu L,et al.Identification and analysis of the culturable bacterium in the feces of captive giant pandas in high-altitude:Taking yunnan wildlife park in kunming in spring for example[J].Sichuan Journal of Zoology,2015,34(6):852-858(in Chinese)周杰珑,李非平,刘丽,等.高海拔地区圈养大熊猫粪便可培养细菌分析——以云南野生动物园春季为例[J].四川动物,2015,34(6):852-858
[13]Suau A,Bonnet R,Sutren M,et al.Direct analysis of genes encoding 16S r RNA from complex communities reveals many novel molecular species within the human gut[J].Applied&Environmental Microbiology,1999,65(11):4799-4807
[14]Gao L,Xiong HS,Peng L,et al.Application of some culture-Independent detection approaches based on 16S r RNA in the research of gastrointestinal tract microbiota[J].Chinese Journal of Animal Science,2008,44(15):56-60(in Chinese)高岚,熊浩山,彭莉,等.以16S r RNA为基础的检测技术在肠道菌群研究中的应用[J].中国畜牧杂志,2008,44(15):56-60
[15]Wang XY,Yuan T,Liao H,et al.Construction of the 16S r DNA clone library of intestinal flora of the captive elderly giant panda[J].China Animal Husbandry&Veterinary Medicine,2015,42(6):1402-1408(in Chinese)王晓艳,袁听,廖虹,等.圈养老年大熊猫肠道菌群16S r DNA克隆文库的建立[J].中国畜牧兽医,2015,42(6):1402-1408
[16]Wei FW,Wang X,Wu Q.The giant panda gut microbiome[J].Trends in Microbiology,2015,23(8):450-452
[17]Sch?fer J,J?ckel U,K?mpfer P.Development of a new PCR primer system for selective amplification of Actinobacteria[J].FEMS Microbiology Letters,2010,311(2):103-112
[18]Li XL,Gu YF,Zhang XP,et al.Nitrobacteria community and diversity in the rhizosphere soil of mature tobacco[J].Scientia Agricultura Sinica,2011,44(12):2462-2468(in Chinese)李小林,辜运富,张小平,等.烟草成熟期根际硝化细菌种群的结构及其多样性[J].中国农业科学,2011,44(12):2462-2468
[19]Sanguinetti CJ,Dias Neto E,Simpson A.Rapid silver staining and recovery of PCR products separated on polyacrylamide gels[J].Biotechniques,1994,17(5):914-921
[20]Eichner CA,Erb RW,Timmis KN,et al.Thermal gradient gel electrophoresis analysis of bioprotection from pollutant shocks in the activated sludge microbial community[J].Applied&Environmental Microbiology,1999,65(1):102-109
[21]Larkin MA,Blackshields G,Brown NP,et al.Clustal W and Clustal X version 2.0[J].Bioinformatics,2007,23(21):2947-2948
[22]Tamura K,Peterson D,Peterson N,et al.MEGA 5:molecular evolutionary genetics analysis using maximum likelihood,evolutionary distance,and maximum parsimony methods[J].Molecular Biology and Evolution,2011,28(10):2731-2739
[23]?milauer P,Lep?J.Multivariate Analysis of Ecological Data Using CANOCO 5[M].Cambridge:Cambridge University Press,2003
[24]Gu YF,Zhang XP,Tu SH,et al.Soil microbial biomass,crop yields,and bacterial community structure as affected by long-term fertilizer treatments under wheat-rice cropping[J].European Journal of Soil Biology,2009,45(3):239-246
[25]Ruan JS.Bergey’s Manual of Systematic Bacteriology(Second Edition)Volume 5 and the study of Actinomycetes systematic in China[J].Acta Microbiologica Sinica,2013,53(6):521-530(in Chinese)阮继生.“伯杰氏系统细菌学手册(第二版)”第5卷与我国的放线菌系统学研究[J].微生物学报,2013,53(6):521-530
[26]Kim TK,Garson MJ,Fuerst JA.Marine actinomycetes related to the‘Salinospora’group from the Great Barrier Reef sponge Pseudoceratina clavata[J].Environmental Microbiology,2005,7(4):509-518
[27]Ward AC,Bora N.Diversity and biogeography of marine actinobacteria[J].Current Opinion in Microbiology,2006,9(3):279-286
[28]Zhang HT,Lee YK,Zhang W,et al.Culturable actinobacteria from the marine sponge Hymeniacidon perleve:isolation and phylogenetic diversity by 16S r RNA gene-RFLP analysis[J].Antonie van Leeuwenhoek,2006,90(2):159-169
[29]Zhao HX.Study on the diversity and function of intestinal bacteria flora from different wild animals[D].Changchun:Master’s Thesis of Jilin Agricultural University,2015(in Chinese)赵晗旭.不同野生动物肠道微生物多样性分析及功能初步研究[D].长春:吉林农业大学硕士学位论文,2015
[30]Zeng HY.Diversity of symbiotic anctinobacteria from healthy ruminant faeces[D].Guangzhou:Master’s Thesis of Sun Yat-Sen University,2012(in Chinese)曾华颖.反刍动物肠道共生放线菌类群多样性分析[D].广州:中山大学硕士学位论文,2012
[31]Tan HM,Deng QL,Cao LX.Ruminant feces harbor diverse uncultured symbiotic actinobacteria[J].World Journal of Microbiology and Biotechnology,2014,30(3):1093-1100
[32]Jiang Y,Cao YR,Cai XF,et al.Diversity and bioactivities of cultured actinomycete isolated from animal feces and Xylocopa dissimilis[J].Acta Microbiologica Sinica,2009,49(9):1152-1157(in Chinese)姜怡,曹艳茹,蔡祥凤,等.三种动物粪便及一种虫体可培养放线菌的多样性及其生物活性[J].微生物学报,2009,49(9):1152-1157
[33]Essoussi I,Ghodhbane-Gtari F,Amairi H,et al.Esterase as an enzymatic signature of Geodermatophilaceae adaptability to Sahara desert stones and monuments[J].Journal of Applied Microbiology,2010,108(5):1723-1732
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