基于MiSeq测序技术分析红鳍东方鲀养殖环境菌群多样性
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摘要
养殖环境中的微生物在物质循环及能量流动中发挥着重要的作用。对养殖环境中微生物多样性研究,不仅可以为养殖过程中病害防治提供基础,还可以发现新的微生物资源。本文利用Mi Seq测序技术分析了大连某养殖公司红鳍东方鲀养殖环境中室内养殖环境水、室外入水口水和底泥中菌群结构特征,从室内养殖环境水、室外入水口水和室外入水口底泥样品中分别获得27164、41146和30474条有效序列。结果表明:红鳍东方鲀养殖环境中具有较高的菌群多样性,其中室外入水口底泥中菌群多样性较其相应养殖水环境中菌群多样性高;3个样品中的细菌可归为33个门类,除常见报道的绿弯菌门、拟杆菌门、浮霉菌门、蓝藻门、酸杆菌门、变形菌门、梭杆菌门、疣微菌门、厚壁菌门和放线菌门外,还有23个鲜见报道的门类的菌群被检测到;各样品中菌群组成虽有差异,但变形菌门和拟杆菌门为优势菌,约占68.33%~93.37%。本文揭示了大连某养殖公司红鳍东方鲀池塘养殖环境中的菌群多样性和丰度,比较了其菌群结构及差异,为红鳍东方鲀健康养殖提供参考。
Microorganisms play an important role in matter cycle and energy flow in aquaculture environment. Studies on microorganism diversity in aquaculture environment not only lay the foundation of disease control but also explore new microorganism resources. In this study,Illumina Miseq was used to analyze the structure characters of microbiome living in Dalian Takifugu rubripes-culturing water indoors,water inlet point outdoors and bottom sludge respectively. For the three samples,27164,41146 and 30474 effective sequences were selected and utilized to perform bacterial diversity analysis. The results disclosed great bacterial diversities in the Takifugu rubripes aquaculture environment. Furthermore,the richness and diversity indices in the sediment samples were higher than that in the water samples. In total,33 phyla were identified. In addition to Chloroflexi,Bacteroidetes,Planctomycetes,Cyanophyta,Acidobacteria,Proteobacteria,Fusobacterium,Verrucomicrobia,Firmicutes and Actinobacteria that have often been reported,other 23 phyla rarely reported were detected too. Although there were differences in microbial composition between these three samples,Proteobacteria and Bacteroidetes( 68.33%- 93.37%)were the dominant groups in all samples. This study reveals the bacterial diversity and abundance in Dalian Takifugu rubripes culturing environment,compares the bacterial community structures and provides guidance for healthy aquaculture of Takifugu rubripes.
Microorganisms play an important role in matter cycle and energy flow in aquaculture environment. Studies on microorganism diversity in aquaculture environment not only lay the foundation of disease control but also explore new microorganism resources. In this study,Illumina Miseq was used to analyze the structure characters of microbiome living in Dalian Takifugu rubripes-culturing water indoors,water inlet point outdoors and bottom sludge respectively. For the three samples,27164,41146 and 30474 effective sequences were selected and utilized to perform bacterial diversity analysis. The results disclosed great bacterial diversities in the Takifugu rubripes aquaculture environment. Furthermore,the richness and diversity indices in the sediment samples were higher than that in the water samples. In total,33 phyla were identified. In addition to Chloroflexi,Bacteroidetes,Planctomycetes,Cyanophyta,Acidobacteria,Proteobacteria,Fusobacterium,Verrucomicrobia,Firmicutes and Actinobacteria that have often been reported,other 23 phyla rarely reported were detected too. Although there were differences in microbial composition between these three samples,Proteobacteria and Bacteroidetes( 68.33%- 93.37%)were the dominant groups in all samples. This study reveals the bacterial diversity and abundance in Dalian Takifugu rubripes culturing environment,compares the bacterial community structures and provides guidance for healthy aquaculture of Takifugu rubripes.
引文
白洁,李海艳,赵阳国.2009.黄海北部不同站位海洋细菌群落分布特征.微生物学报,49(3):343-350.
陈永伟,栾鑫,段继周,等.2014.青岛中港海水全浸初期不锈钢表面生物膜的细菌多样性分析.海洋与湖沼,45(5):1064-1070.
董逸.2013.我国黄、东海典型海域微生物群落结构及其与环境变化的关系(博士学位论文).青岛:中国科学院海洋研究所.
段曌,肖炜,王永霞,等.2011.454测序技术在微生物生态学研究中的应用.微生物学杂志,31(5):76-81.
李学恭,徐俊,肖湘.2013.深海微生物高压适应与生物地球化学循环.微生物学通报,40(1):59-70.
李雪松,梁君荣,陈长平,等.2006.泉州湾虾池浮游植物种类多样性研究.厦门大学学报,45(S1):234-249.
刘军,张帅帅,杨娜,等.2010.北极海泥菌群的分离鉴定及生物学特性的初步研究.生物技术通报,(1):136-141.
马海燕.2007.刺参(Stichopus japonicus)烂皮病病灶组织显微观察及病原的初步研究(硕士学位论文).青岛:中国海洋大学.
乔迁,韩娜娜,陈超,等.2010.致病性假交替单胞菌的分离鉴定及药敏特性研究.安徽农业科学,38(26)14224-14226.
任欣欣,姜昊,冷欣,等.2013.蓝藻胞外多糖的生态学意义及其工业应用.生态学杂志,32(3):762-771.
王奎旗,陈梅,高天翔.2001.东方鲀属鱼类的分类与区系分布研究.青岛海洋大学学报,31(6):855-860.
王轶南,朱世伟,常亚青.2010.刺参肠道及养殖池塘菌群组成的PCR-DGGE指纹图谱分析.渔业科学进展,31(3):119-122.
徐姗楠,林华剑,戴明,等.2014.广西近岸海域浮游植物群落的生态特征.生态学杂志,33(10):2733-2739.
徐晓宇,刘和.2010.454测序法在环境微生物生态研究中的应用.生物技术通报,(1):73-77.
郑宗华,高铭标,冯良利,等.1988.红鳍东方鲀人工育苗技术探讨.福建水产,(3):18-23.
周林文.2011.MiS eq:新一代个人化测序仪.生物技术世界,(10):14-16.
Amann RI,Ludwig W,Schleifer KH.1995.Phylogenetic identification and in situ detection of individual microbial cells\swithout cultivation.Microbiological Reviews,59:143-169.
Bell TH,Yergeau E,Maynard C,et al.2013.Predictable bacterial composition and hydrocarbon degradation in Arctic\ssoils following diesel and nutrient disturbance.The ISMEJournal,7:1200-1210.
Edgar RC,Haas BJ,Clemente JC,et al.2011.UCHIME improves sensitivity and speed of chimera detection.Bioinformatics,27:2194-2200.
Feller G,Narinx E,Arpigny JL,et al.1996.Enzymes from psychrophilic organisms.FEMS Microbiology Reviews,18:189-202.
Gosalbes MJ,Durban A,Pignatelli M,et al.2011.Metatranscriptomic approach to analyze the functional human gut\smicrobiota.PLoS ONE,6:e17447.
Holmstrom C,Kjellebery S.1999.Marine Pseudoalteromonas\sspecies are associated with higher organisms and produce\sbiologically active extracellular agents.FEMS Microbiology\sEcology,30:285-293.
Hou YH,Wang TH,Long H,et al.2006.Novel cold-adaptive\sPenicillium strain FS010 secreting thermo-labile xylanase\sisolated from Yellow Sea.Acta Biochimicaet Biophysica\sSinica,38:142-149.
Huber I,Spanggaard B,Appel KF,et al,2004.Phylogenetic\sanalysis and in situ identification of the intestinal microbial\scommunity of rainbow trout(Oncorhynchus mykiss,Walbaum).Journal of Applied Microbiology,96:117-132.
Li L,Kato C,Horikoshi K.1999.Microbial diversity in sediments collected from the deepest cold-seep area,the Japan\sTrench.Marine Biotechnology,1:391-400.
Lozupone CA,Knight R,2007.Global patterns in bacterial\sdiversity.Proceedings of the National Academy of Sciences\sof the United States of America,104:11436-11440.
Markle JGM,Frank DN.2013.Sex differences in the gut microbiome drive hormone-dependent regulation of autoimmunity.Science,339:1084-1088.
McC aig AE,Glover LA,Prosser JI.1999.Molecular analysis of\sbacterial community structure and diversity in unimproved\sand improved upland grass pastures.Applied and Environmental Microbiology,65:1721-1730.
Michand L,Lo Gindice A,Troussellier M,et al.2009.Phylogenetic characterization of the heterotrophic bacterial communities inhabiting a marine recirculating aquaculture system.Journal of Applied Microbiology,107:1935-1946.
Oikonomou G,Machado VS,Santisteban C,et al.2012.Microbial diversity of bovine mastitic milk as described by pyrosequencing of metagenomic 16S rD NA.PLoS ONE,7:e47671.
Payne MS,Hall MR,Bannister R,et al.2006.Microbial diversity within the water column of a larval rearing system for\sthe ornate rock lobster Panulirus ornatus.Aquaculture,258:80-90.
Pop M,Phillippy A,Delcher AL,et al.2004.Comparative genome assembly.Briefings in Bioinformatics,5:237-248.
Rosselló-Mora R,Thamdrup B,Schfer H,et al.1999.The response of the microbial community of marine sediments to\sorganic carbon input under anaerobic conditions.Systematic\sand Applied Microbiology,22:237-248.
Rutvisuttinunt W,Chinnawirotpisan P,Simasathien S,et al.2013.Simultaneous and complete genome sequencing of influenza a and b with high coverage by Illumina MiS eq Platform.Journal of Virological Methods,193:394-404.
Schloss PD,Westcott SL,Ryabin T,et al.2009.Introducing\smothur:Open-source,platform-independent,communitysupported software for describing and comparing microbial\scommunities.Applied and Environmental Microbiology,75:7537-7541.
Shiina A,Itoi S,Washio S,et al.2006.Molecular identification\sof intestinal microflora in Takifugu niphobles.Comparative\sBiochemistry&Physiology Part D Genomics&Proteomics,1:128-132.
Shendure J,Ji H.2008.Next-generation DNA sequencing.Nature Biotechnology,26:1135-1145.
Simpson JT,Wong K,Jackman SD,et al.2009.AByS S:A parallel assembler for short read sequence data.Genome\sResearch,19:1117-1123.
Sukui M,Nakagawa Y,Harayama S,et al.2001.Phylogenetic\sanalysis and taxonomic study of marine Cytophaga-like bacteria:Proposal for Tenacibaculum gen.nov.with Tenacibaculum maritimum comb.nov.and Tenacibaculum ovolyticum\scomb.nov.,and description of Tenacibaculum mesophilum\ssp.nov.and Tenacibaculum amylolyticum sp.Nov.International Journal of Systematic and Evolutionary Microbiology,51:1639-1652.
Thompson JR,Pacocha S,Pharino C,et al.2005.Genotypic\sdiversity within a natural coastal bacterioplankton population.Science,307:1311-1313.
Williams ST,Foster PG,Littlewood DT.2014.The complete\smitochondrial genome of a turbinid vetigastropod from\sMiS eq Illumina sequencing of genomic DNA and steps towards a resolved gastropod phylogeny.Gene,533:38-47.
Zerbino DBE.2008.Velvet:Algorithms for de novo short read\sassembly using de Bruijn graphs.Genome Research,18:821-829.
陈永伟,栾鑫,段继周,等.2014.青岛中港海水全浸初期不锈钢表面生物膜的细菌多样性分析.海洋与湖沼,45(5):1064-1070.
董逸.2013.我国黄、东海典型海域微生物群落结构及其与环境变化的关系(博士学位论文).青岛:中国科学院海洋研究所.
段曌,肖炜,王永霞,等.2011.454测序技术在微生物生态学研究中的应用.微生物学杂志,31(5):76-81.
李学恭,徐俊,肖湘.2013.深海微生物高压适应与生物地球化学循环.微生物学通报,40(1):59-70.
李雪松,梁君荣,陈长平,等.2006.泉州湾虾池浮游植物种类多样性研究.厦门大学学报,45(S1):234-249.
刘军,张帅帅,杨娜,等.2010.北极海泥菌群的分离鉴定及生物学特性的初步研究.生物技术通报,(1):136-141.
马海燕.2007.刺参(Stichopus japonicus)烂皮病病灶组织显微观察及病原的初步研究(硕士学位论文).青岛:中国海洋大学.
乔迁,韩娜娜,陈超,等.2010.致病性假交替单胞菌的分离鉴定及药敏特性研究.安徽农业科学,38(26)14224-14226.
任欣欣,姜昊,冷欣,等.2013.蓝藻胞外多糖的生态学意义及其工业应用.生态学杂志,32(3):762-771.
王奎旗,陈梅,高天翔.2001.东方鲀属鱼类的分类与区系分布研究.青岛海洋大学学报,31(6):855-860.
王轶南,朱世伟,常亚青.2010.刺参肠道及养殖池塘菌群组成的PCR-DGGE指纹图谱分析.渔业科学进展,31(3):119-122.
徐姗楠,林华剑,戴明,等.2014.广西近岸海域浮游植物群落的生态特征.生态学杂志,33(10):2733-2739.
徐晓宇,刘和.2010.454测序法在环境微生物生态研究中的应用.生物技术通报,(1):73-77.
郑宗华,高铭标,冯良利,等.1988.红鳍东方鲀人工育苗技术探讨.福建水产,(3):18-23.
周林文.2011.MiS eq:新一代个人化测序仪.生物技术世界,(10):14-16.
Amann RI,Ludwig W,Schleifer KH.1995.Phylogenetic identification and in situ detection of individual microbial cells\swithout cultivation.Microbiological Reviews,59:143-169.
Bell TH,Yergeau E,Maynard C,et al.2013.Predictable bacterial composition and hydrocarbon degradation in Arctic\ssoils following diesel and nutrient disturbance.The ISMEJournal,7:1200-1210.
Edgar RC,Haas BJ,Clemente JC,et al.2011.UCHIME improves sensitivity and speed of chimera detection.Bioinformatics,27:2194-2200.
Feller G,Narinx E,Arpigny JL,et al.1996.Enzymes from psychrophilic organisms.FEMS Microbiology Reviews,18:189-202.
Gosalbes MJ,Durban A,Pignatelli M,et al.2011.Metatranscriptomic approach to analyze the functional human gut\smicrobiota.PLoS ONE,6:e17447.
Holmstrom C,Kjellebery S.1999.Marine Pseudoalteromonas\sspecies are associated with higher organisms and produce\sbiologically active extracellular agents.FEMS Microbiology\sEcology,30:285-293.
Hou YH,Wang TH,Long H,et al.2006.Novel cold-adaptive\sPenicillium strain FS010 secreting thermo-labile xylanase\sisolated from Yellow Sea.Acta Biochimicaet Biophysica\sSinica,38:142-149.
Huber I,Spanggaard B,Appel KF,et al,2004.Phylogenetic\sanalysis and in situ identification of the intestinal microbial\scommunity of rainbow trout(Oncorhynchus mykiss,Walbaum).Journal of Applied Microbiology,96:117-132.
Li L,Kato C,Horikoshi K.1999.Microbial diversity in sediments collected from the deepest cold-seep area,the Japan\sTrench.Marine Biotechnology,1:391-400.
Lozupone CA,Knight R,2007.Global patterns in bacterial\sdiversity.Proceedings of the National Academy of Sciences\sof the United States of America,104:11436-11440.
Markle JGM,Frank DN.2013.Sex differences in the gut microbiome drive hormone-dependent regulation of autoimmunity.Science,339:1084-1088.
McC aig AE,Glover LA,Prosser JI.1999.Molecular analysis of\sbacterial community structure and diversity in unimproved\sand improved upland grass pastures.Applied and Environmental Microbiology,65:1721-1730.
Michand L,Lo Gindice A,Troussellier M,et al.2009.Phylogenetic characterization of the heterotrophic bacterial communities inhabiting a marine recirculating aquaculture system.Journal of Applied Microbiology,107:1935-1946.
Oikonomou G,Machado VS,Santisteban C,et al.2012.Microbial diversity of bovine mastitic milk as described by pyrosequencing of metagenomic 16S rD NA.PLoS ONE,7:e47671.
Payne MS,Hall MR,Bannister R,et al.2006.Microbial diversity within the water column of a larval rearing system for\sthe ornate rock lobster Panulirus ornatus.Aquaculture,258:80-90.
Pop M,Phillippy A,Delcher AL,et al.2004.Comparative genome assembly.Briefings in Bioinformatics,5:237-248.
Rosselló-Mora R,Thamdrup B,Schfer H,et al.1999.The response of the microbial community of marine sediments to\sorganic carbon input under anaerobic conditions.Systematic\sand Applied Microbiology,22:237-248.
Rutvisuttinunt W,Chinnawirotpisan P,Simasathien S,et al.2013.Simultaneous and complete genome sequencing of influenza a and b with high coverage by Illumina MiS eq Platform.Journal of Virological Methods,193:394-404.
Schloss PD,Westcott SL,Ryabin T,et al.2009.Introducing\smothur:Open-source,platform-independent,communitysupported software for describing and comparing microbial\scommunities.Applied and Environmental Microbiology,75:7537-7541.
Shiina A,Itoi S,Washio S,et al.2006.Molecular identification\sof intestinal microflora in Takifugu niphobles.Comparative\sBiochemistry&Physiology Part D Genomics&Proteomics,1:128-132.
Shendure J,Ji H.2008.Next-generation DNA sequencing.Nature Biotechnology,26:1135-1145.
Simpson JT,Wong K,Jackman SD,et al.2009.AByS S:A parallel assembler for short read sequence data.Genome\sResearch,19:1117-1123.
Sukui M,Nakagawa Y,Harayama S,et al.2001.Phylogenetic\sanalysis and taxonomic study of marine Cytophaga-like bacteria:Proposal for Tenacibaculum gen.nov.with Tenacibaculum maritimum comb.nov.and Tenacibaculum ovolyticum\scomb.nov.,and description of Tenacibaculum mesophilum\ssp.nov.and Tenacibaculum amylolyticum sp.Nov.International Journal of Systematic and Evolutionary Microbiology,51:1639-1652.
Thompson JR,Pacocha S,Pharino C,et al.2005.Genotypic\sdiversity within a natural coastal bacterioplankton population.Science,307:1311-1313.
Williams ST,Foster PG,Littlewood DT.2014.The complete\smitochondrial genome of a turbinid vetigastropod from\sMiS eq Illumina sequencing of genomic DNA and steps towards a resolved gastropod phylogeny.Gene,533:38-47.
Zerbino DBE.2008.Velvet:Algorithms for de novo short read\sassembly using de Bruijn graphs.Genome Research,18:821-829.