琼东海域珊瑚共附生放线菌的多样性
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摘要
应用免培养方法与可培养方法相结合的方式对琼东海域6种珊瑚样品中的共附生放线菌进行研究,具体为滨珊瑚(Porite)、扁脑珊瑚(Platygyra)、角蜂巢珊瑚(Favites)、盔形珊瑚(Galaxea)和鹿角珊瑚(Acropora).免培养方法中,利用放线菌特异性引物对样品总DNA进行16S rRNA基因序列扩增,并将扩增产物进行Miseq高通量测序,测序结果分析表明,6种珊瑚样品中的放线菌序列属于37个分类单元,主要分布在酸微菌目、棒杆菌目、微球菌目、丙酸杆菌目和假诺卡式菌目,其中37.8%的分类单元与已有效发表菌株的序列相似性小于97%,可能代表着放线菌新的类群.可培养方法中,通过6种培养基分得137株放线菌,分属于放线菌门放线菌纲5个目11个科下的20个属,优势类群为分枝杆菌属(Mycobacterium)、考克氏菌属(Kocuria)和微球菌属(Micrococcus),其中放线孢菌属(Actinomycetospora)、柠檬球菌属(Citricoccus)和涅斯捷连科氏菌属(Nesterenkonia)是第一次从珊瑚样品中发现;琼东海域珊瑚共附生放线菌多样性丰富,新类型的放线菌资源挖掘潜力比较大.
In this study,six coral samples belonging to the genera Porite,Platygyra,Favites,Galaxea,Acropora and Favia were sampled from the east coast of Hainan Island. Diversity of coral-associated actinobacteria was investigated using culture-dependent and culture-independent approaches. Specific primers for the phylum Actinobacteria were used to amplify the actinobacterial 16 S rRNA gene,and 16 S rRNA gene libraries were constructed and sequenced with the Illumina Mi Seq platform. The analysis of 16 S rRNA gene sequences showed that the actinobacterial sequences were grouped in 37 OTUs on the basis of 97% sequence similarity within one OTU. These OTUs mainly belonged to classes Acidimicrobiales,Corynebacteriales,Micrococcales,Propionibacteriales and Pseudonocardiales. 37. 8 % of these OTUs showed less than 97% 16 S rRNA gene sequence similarity with validly published species and represented novel taxa. A total of 137 strains were isolated used six different media. As results,they belong to 20 known genera,among them the genera Mycobacterium,Kocuria and Micrococcus were the dominants.The genera Actinomycetospora,Citricoccus and Nesterenkonia were first isolated from coral hosts. Cumulatively,the east coast of Hainan Island harbors abundant actinobacteria,including large number of unknown actinobacterial groups.
In this study,six coral samples belonging to the genera Porite,Platygyra,Favites,Galaxea,Acropora and Favia were sampled from the east coast of Hainan Island. Diversity of coral-associated actinobacteria was investigated using culture-dependent and culture-independent approaches. Specific primers for the phylum Actinobacteria were used to amplify the actinobacterial 16 S rRNA gene,and 16 S rRNA gene libraries were constructed and sequenced with the Illumina Mi Seq platform. The analysis of 16 S rRNA gene sequences showed that the actinobacterial sequences were grouped in 37 OTUs on the basis of 97% sequence similarity within one OTU. These OTUs mainly belonged to classes Acidimicrobiales,Corynebacteriales,Micrococcales,Propionibacteriales and Pseudonocardiales. 37. 8 % of these OTUs showed less than 97% 16 S rRNA gene sequence similarity with validly published species and represented novel taxa. A total of 137 strains were isolated used six different media. As results,they belong to 20 known genera,among them the genera Mycobacterium,Kocuria and Micrococcus were the dominants.The genera Actinomycetospora,Citricoccus and Nesterenkonia were first isolated from coral hosts. Cumulatively,the east coast of Hainan Island harbors abundant actinobacteria,including large number of unknown actinobacterial groups.
引文
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[29]Reshef L,Koren O,Loya Y,et al.The Coral Probiotic Hypothesis[J].Environmental Microbiology,2006,8(12):2 068-2 073.
[2]Mahmoud H M,Kalendar A A.Coral-associated actinobacteria:diversity,abundance,and biotechnological potentials[J].Front Microbiol,2016,7:204.
[3]Kuang W,Li J,Zhang S,et al.Diversity and distribution of actinobacteria associated with reef coral Porites lutea[J].Front Microbiol,2015,6:1 094.
[4]周俊勇.南海珊瑚礁样品中菌株分离及其活性代谢产物研究[D].福州:福建师范大学,2011.
[5]Harvell C D,Kim K,Burkholder J M,et al.Emerging marine diseases-climate links and anthropogenic factors[J].Science,1999,285(5 433):1 505-1 510.
[6]Schloss P D,Westcott S L,Ryabin T,et al.Introducing mothur:open-source,platform-independent,community-supported software for describing and comparing microbial communities[J].Applied and Environmental Microbiology,2009,75(23):7 537-7 541.
[7]Giovannoni S J,Mullins T D,and Field K G.Microbial diversity in oceanic systems:rRNA approaches to the study of unculturable microbes[M].Berlin Heidelberg:Joint I.Springer Berlin Heidelberg,1995:217-248.
[8]Chao A.Nonparametric estimation of the number of classes in a population[J].Scandinavian Journal of Statistics,1984,11(4):265-270.
[9]Wang Y,Sheng H F,He Y,et al.Comparison of the levels of bacterial diversity in freshwater,intertidal wetland,and marine sediments by using millions of illumina tags[J].Applied and Environmental Microbiology,2012,78(23):8 264-8 271.
[10]Kim O,Cho Y,Lee K,et al.Introducing Ez Taxon-e:a prokaryotic 16S rRNA gene sequence database with phylotypes that represent uncultured species[J].International Journal of Systematic and Evolutionary Microbiology,2012,62(3):716-721.
[11]Tamura K,Peterson D,Peterson N,et al.MEGA5:Molecular evolutionary genetics analysis using maximum likelihood,evolutionary distance,and maximum parsimony methods[J].Molecular Biology and Evolution,2011,28(10):2 731-2 739.
[12]Kemp P F,Aller J Y.Bacterial diversity in aquatic and other environments:what 16S r DNA libraries can tell us[J].FEMS Microbiol Ecology,2004,47(2):161-177.
[13]Simu K,Hagstr9m K.Oligotrophic bacterioplankton with a novel single-cell life strategy[J].Applied and Environmental Microbiology,2004,70(4):2 445-2 451.
[14]Xiong Z Q,Liu Q X,Pan Z L,et al.Diversity and bioprospecting of culturable actinomycetes from marine sediment of the Yellow Sea,China[J].Arch Microbiol,2015,197(2):299-309.
[15]Zhang G,Cao T,Ying J,et al.Diversity and novelty of actinobacteria in Arctic marine sediments[J].Antonie Van Leeuwenhoek,2014,105(4):743-754.
[16]Sun W,Dai S,Jiang S,et al.Culture-dependent and culture-independent diversity of Actinobacteria associated with the marine sponge Hymeniacidon perleve from the South China Sea[J].Antonie Van Leeuwenhoek,2010,98(1):65-75.
[17]Stach J E M,Maldonado L A,Ward A C,et al.New primers for the class Actinobacteria:application to marine and terrestrial environments[J].Environmental Microbiology,2003,5(10):828-841.
[18]Li J,Dong J D,Yang J,et al.Detection of polyketide synthase and nonribosomal peptide synthetase biosynthetic genes from antimicrobial coral-associated actinomycetes[J].Antonie Van Leeuwenhoek,2014,106(4):623-635.
[19]De-Voogd N J,Cleary D F R,Polónia A R M,et al.Bacterial community composition and predicted functional ecology of sponges,sediment and seawater from the thousand islands reef complex,West Java,Indonesia[J].FEMS Microbiol Ecology,2015,91(4),doi:10.1093/femsec/fivo19.
[20]Elahwany A M D,Ghozlan H A,Elsharif H A,et al.Phylogenetic diversity and antimicrobial activity of marine bacteria associated with the soft coral Sarcophyton glaucum[J].Journal of Basic Microbiology,2015,55(1):2-10.
[21]Sarmiento-Vizcaíno A,González V,Bra1a A F,et al.Pharmacological potential of phylogenetically diverse actinobacteria isolated from deep-sea coral ecosystems of the submarine Avilés Canyon in the Cantabrian Sea[J].Microbial Ecology,2017:73(2):338-352.
[22]Pham T M,Wiese J,Wenzel-Storjohann A,et al.Diversity and antimicrobial potential of bacterial isolates associated with the soft coral Alcyonium digitatum from the Baltic Sea[J].Antonie Van Leeuwenhoek,2016,109(1):105-119.
[23]陈淇,龙丽娟,张偲,等.澄黄滨珊瑚和丛生盔型珊瑚非培养放线菌多样性[J].微生物学通报,2014,41(4):691-698.
[24]马亮,张文军,朱义广,等.永兴岛白穗软珊瑚共附生放线菌筛选及部分活性次级代谢产物的鉴定[J].微生物学报,2013,53(10):1 063-1 071.
[25]Hong M J,Yu Y T,Chen C A,et al.Influence of species specificity and other factors on bacteria associated with the coral Stylophora pistillata in Taiwan[J].Applied and Environmental Microbiology,2009,75(24):7 797-7 806.
[26]Littman R A,Willis B L,Pfeffer C,et al.Diversities of coral-associated bacteria differ with location,but not species,for three acroporid corals on the Great Barrier Reef[J].FEMS Microbiol Ecology,2009,68(2):152-163.
[27]Rohwer F.Diversity and distribution of coral-associated bacteria[J].Marine Ecology Progress Series,2002,243(4):1-10.
[28]Kvennefors E C E,Sampayo E,Ridgway T,et al.Bacterial communities of two ubiquitous Great Barrier reef corals reveals both site-and species-specificity of common bacterial associates[J].PLo S One,2010,5(4):e10401.
[29]Reshef L,Koren O,Loya Y,et al.The Coral Probiotic Hypothesis[J].Environmental Microbiology,2006,8(12):2 068-2 073.