新疆乌鲁木齐10号冷泉古菌群落结构多样性
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摘要
【目的】探究新疆地震断裂带含硫冷泉泉水古菌群落组成及多样性。【方法】利用酶解法直接从冷泉泉水样品中提取环境总DNA,采用古菌通用引物对16S rRNA基因进行扩增,构建16S rRNA基因克隆文库,通过Alu I和Afa I两种限制性内切酶对随机挑选的115个阳性克隆子进行酶切分型,将不同酶切带型对应的克隆子送样测序,测序结果与GenBank序列进行比对并构建16S rRNA基因系统发育树。【结果】古菌克隆文库中共得到44个不同的酶切带型,BLAST序列比对和系统发育分析将它们划分于广古菌门(Euryarchaeota,94.78%)和奇古菌门(Thaumarchaeota,4.35%)。奇古菌门克隆与Nitrosopumilus.sp序列相似性达到了93%;而广古菌门类群较为多样,其中,42.61%的克隆子属于RC-V cluster,20.87%与13.91%的克隆子分别属于LDS cluster和Methanomicrobiales,4.35%的克隆子与甲烷厌氧氧化相关的类群(ANME-1a-FW)具有较高的的相似。另外,13.05%的克隆子属于广古菌门中的未知类群。【结论】乌鲁木齐10号泉水体中广古菌类群多样,可能蕴藏有大量潜在的古菌新类群。
[Objective]We explored the composition and diversity of archaea in a cold sulfur spring water in Xinjiang earthquake fault zone.[Methods]Environmental total DNA was extracted directly with enzymatic lysis method from a cold sulfur spring water.We constructed clone library of 16S rRNA gene amplified with archaeal-specific primers.A total of 115 positive clones were selected randomly from the library and identified by restriction length polymorphism(RFLP) with enzyme Alu I and Afa I.The unique RFLP patterns corresponded clones were selected for sequencing,BLAS alignment and constructing 16S rRNA gene phylogenetic tree.[Results]In total,44 operational taxonomic units(OTUs) were determined from the library.BLAST and phylogenetic analysis indicated that these OTUs were affiliated with Euryarchaeota(94.78%) and Thaumarchaeota(4.35%).Only one Thaumarchaeotal clone was detected and most related to the genus Nitrosopumilus with 93% similarity.Euryarchaeotal clones were abundant and diverse.Of them,42.61% of clones belonged to RC-V cluster;13.91% of clones,20.87% of clones were classified into LDS cluster and Methanomicrobiales respectively;4.35% of clones had high similarity with ANME-1a-FW,which were involved in Anaerobic oxidation of methane(AOM).In addition,we also detected some(13.05%) unknown Euryarchaeotal clones.[Conclusion]Euryarchaeota in the environment were diverse,and possibly with a large fraction of potential novel species.
[Objective]We explored the composition and diversity of archaea in a cold sulfur spring water in Xinjiang earthquake fault zone.[Methods]Environmental total DNA was extracted directly with enzymatic lysis method from a cold sulfur spring water.We constructed clone library of 16S rRNA gene amplified with archaeal-specific primers.A total of 115 positive clones were selected randomly from the library and identified by restriction length polymorphism(RFLP) with enzyme Alu I and Afa I.The unique RFLP patterns corresponded clones were selected for sequencing,BLAS alignment and constructing 16S rRNA gene phylogenetic tree.[Results]In total,44 operational taxonomic units(OTUs) were determined from the library.BLAST and phylogenetic analysis indicated that these OTUs were affiliated with Euryarchaeota(94.78%) and Thaumarchaeota(4.35%).Only one Thaumarchaeotal clone was detected and most related to the genus Nitrosopumilus with 93% similarity.Euryarchaeotal clones were abundant and diverse.Of them,42.61% of clones belonged to RC-V cluster;13.91% of clones,20.87% of clones were classified into LDS cluster and Methanomicrobiales respectively;4.35% of clones had high similarity with ANME-1a-FW,which were involved in Anaerobic oxidation of methane(AOM).In addition,we also detected some(13.05%) unknown Euryarchaeotal clones.[Conclusion]Euryarchaeota in the environment were diverse,and possibly with a large fraction of potential novel species.
引文
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[2]Danielopol DL,Griebler C,Gunatilaka A,NotenboomJ.Present state and future prospects for groundwaterecosystems.Environmental Conservation,2003,30(26):104-130.
[3]Gribler C,Lueder T.Microbial biodiversity ingroundwater ecosystems.Freshwater Biology,2009,54(4):1365-2427.
[4]Wall FD,Blackburn TH,Brussaard L,Htuchings P,Palmer MA.Linking biodiversity and ecosystemfunctioning of soils and sediments.Ambiology,1997,26(8):556-562.
[5]Danielopo DL,Pospisil P,Rouch R.Biodiversity ingroundwater:a large scale view.Trends in Ecology andEvolution,2000,15(6):223-224.
[6]Ruth H,Christine M,Thomas A,Christian Rudolph,Robert Huber.New insights into the lifestyle of the cold-loving SM1 euryarchaeon:natural growth as amonospecies biofilm in the subsurface.Applied andEnvironmental Microbiology,2006,72(1):192-199.
[7]Marcus K,Christian R,Christine M,Robert Huber.Acold-loving crenarchaeon is a substantial part of a novelmicrobial community in cold sulphidic marsh water.FEMS Microbiology Ecology,2006,57(1):55-66.
[8]Chaudhary A,Haack SK,Duris JW,March TL.Bacterial and archaeal phylogenetic diversity of a coldsulfur-rich spring on the shoreline of Lake Erie,Michigan.Applied and Environmental Microbiology,2009,75(15):5025-5036.
[9]Nancy NP,Dale TA,Wayne HP,Charel WG,LyleGW.Characterization of the prokaryotic diversity in coldsaline perennial springs of the canadianhigh arctic.Applied and Environmental Microbiology,2007,73(5):1532-1543.
[10]Zeng J,Yang HM,Wu JC,Xu JH,Zhang T,Sun J,Lou K.Uncultured archaeal in cold spring sediment ofShawan,Xinjiang,Chima.Acta Microbiologica Sinica,2010,50(5):574-579.(in Chinese)曾军,杨红梅,吴江超,徐建华,张涛,孙建,娄恺.新疆沙湾冷泉沉积物中免培养古菌多样性初步研究.微生物学报,2010,50(5):574-579.
[11]Li GY,Jiang HC,Hou WG,Wang shang,Huang LQ,Ren HL,Deng SH,Dong HL.Microbial diversity in twocold spring on the Qing hai-Tibetan Plateau.GeoscienceFrontiers,2012,3(3):317-325.
[12]Hu R,Zeng J,Gao XQ,Duan WW,Yang XF,Lou K.Respond of Archaeal Community to HydrogeochemicalElements Changes in the No.Ten Cold Spring of Urumqiin Seismic Fault Zone in Xinjiang.Xin Jiang AgriculturalSciences,2011,48(3):551-556.(in Chinese)胡蓉,曾军,高小其,段魏魏,杨晓芳,娄恺.乌鲁木齐地震断裂带泉水古菌群落对水文地球化学元素的响应.新疆农业科学,2011,48(3):551-556.
[13]Wu JC,Zhang T,Sun J,Shi YW.Functional Diversityof Microbial Community in Seismic Fault Belt Spring ofXinjiang.Xin Jiang Agricultural Sciences,2010,47(5):1052-1056.(in Chinese)吴江超,张涛,孙建,史应武.新疆地震断裂带泉水微生物群落结构功能多样性.新疆农业科学,2010,47(5):1052-1056.
[14]Cui Y,Lan L.Analysis on cause of high mercurycontained in distilled water in Urumqi area.InlandEarthquake,2001,15(4):360-365.(in Chinese)崔勇,兰陵.乌鲁木齐地区汞空白值较高原因分析.内陆地震,2001,15(4):360-365.
[15]Xu LM,Wan QH,Wang D.DNA extraction fromformalin-fixed tissues and genetic diversity of Bai Ji.ActaHydrobiologica Sinica,2005,29(3):272-278.(inChinese)许黎美,万秋红,王丁.福尔马林固定白暨豚标本DNA提取及其遗传多样性的初步研究.水生生物学报,2005,29(3):272-278.
[16]Cui Y,Dilixiati K.Analysis on three long-term trendinganomalies of methane in Urumqi No.10 spring.InlandEarthquake,2004,18(4):371-375.(in Chinese)崔勇,地里夏提.克优木.乌鲁木齐10号泉溶解气甲烷的3次长趋势异常变化的分析.内陆地震,2004,18(4):371-375.
[17]Shaheen B H,Nasreen B,James T H.Depthdistribution of microbial diversity in Mono Lake,ameromictic soda Lake in california.Applied andEnvironmental Microbiology,2003,69(2):1030-1042.
[18]Xu JH,Yang HM,Zeng J,Li Z,Lou K.Bacterialdiversity in a hot gas spring soil of MaNasi CountyXinjiang by culture-independent approach.ActaMicrobiologica Sinica,2010,50(5):568-573.(inChinese)徐建华,杨红梅,曾军,李智,娄恺.新疆玛纳斯热气泉免陪养土壤细菌多样性分析.微生物学报,2010,50(5):568-573.
[19]Fan HP,Xue YF,Zeng Y,Zhou PJ,Ma YH.ArchaealDiversity of ZabuyeLakein Tibet Analyzed by Culture-independent Approach.Acta Microbiologica Sinica,2003,43(4):401-408.(in Chinese)范华鹏,薛燕芬,曾艳,周培谨,马延和.西藏扎布耶茶卡盐碱湖古菌多样性的非培养技术分析.微生物学报,2003,43(4):401-408.
[20]Kimura MA.A simple method for estimating evolutionaryrates of base substitutions through comparative studies ofnucleotide sequences.Journal of Molecular Evolution,1980,16(2):111-120.
[21]Zhang LM,He JZ.A novel archaeal phylum:Thaumarchaeota-A review.Acta Microbiologica Sinica,2012,52(4):411-421.(in Chinese)张丽梅,贺纪正.一个新的古菌类群—奇古菌门(Thaumcrchaeota).微生物学报,2012,52(4):411-421.
[22]Konneke M,Bernhard AE,Torre JR,Jose R,Christopher B,Walker JB,David AS.Isolation of anautrophic ammonia-oxidizing marine archaeon.Nature,2005,437(1038):543-546.
[23]Martens HW,Berube PM,Urakawa H,Jose R,DavidAS.Ammonia oxidation kinetics determine nicheseparation of nitrifying archaea and bacteria.Nature,2009,461(7266):976-979.
[24]Glissman K,Chin KJ,Casper P,Conrad R.Methanogenic pathway and archaeal community structurein the sediment of eutrophic Lake Dagow:effect oftemperature.Microbial Ecology,2004,48(3):389-399.
[25]Grosskopf R,Stubner S,Liesack W.Noveleuryarchaeotal lineages detected on rice roots and intheanoxic bulk soil of flooded rice microcosms.Appliedand Environmental Microbiology,1998,64(12):4983-4989.
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