富营养化湖泊武汉东湖沉积物细菌多样性和系统发育研究

华中科技大学硕士学位论文富营养化湖泊武汉东湖沉积物细菌多样性和系统发育研究姓名：程占冰申请学位级别：硕士专业：生物化学与分子生物学指导教师：杨江科2011-01-21I华中科技大学硕士学位论文摘要水体富营养化已成为严重影响我国生态安全、制约社会经济发展、降低居民生活质量的一大因素。微生物是富营养化水体的分解者，参与了水体氮磷等元素的代谢循环，然而国内关于富营养化水体微生物多样性的分子生态学研究报道尚少，特定功能微生物群落多样性研究更少，因此富营养化水体细菌总群落及具有特定功能微生物群落多样性研究具有重要意义。武汉东湖为一典型富营养化水体，本文以东湖沉积物为研究对象，9月份(2009年)在其4个子湖中心(郭郑湖、汤菱湖、团湖、庙湖)利用5点取样法采集沉积物样品。通过构建16SrRNA基因和亚硝酸还原酶基因(nirS)克隆文库、限制性片段长度多态性分析(RFLP)技术和测序分析等分子生物学手段，初步探讨了环境因素与对微生物群落分布的影响，重点考察了沉积物中细菌群落组成、结构和系统发育，并研究了在氮循环过程中具有主导作用的反硝化细菌微生物多样性。主要研究结果如下：1.关于富营养化湖泊沉积物细菌多样性和系统发育分析(1)环境因子分析表明，T样点所测定各类参数均昀低；M样点污染昀为严重，具有昀高的TN和NH4+浓度；G7和TL样点相似。DOTUR分析表明，在T样点，微生物具有昀高的多样性及丰富度指数，M样点与G7样点相似，丰富度及多样性指数昀低。TN和NH4+浓度可能是影响细菌微生物总群落结构及多样性的重要因素之一。(2)16SrRNA基因系统发育分析表明，该生境具有丰富的物种多样性和种群结构。沉积物细菌主要涵盖9个类群：变形菌门(Proteobacteria)、酸杆菌门(Acidobacteria)、拟杆菌门(Bacteroidetes)、疣微菌门(Verrucomicrobia)及浮霉菌门(Planctomycetes)在4个采样点样品中都有发现；绿弯菌门(Chloroflexi)和厚壁菌门(Firmicutes)只在T和M两个采样点样品中发现，泉古生菌(Crenarchaeota)和广古生菌(Euryarchaeota)，只在M采样点检测到。计数统计表明，优势微生物种群依次为：δ-变形菌亚门(δ-Proteobacteria)、β-变形菌亚门(β-Proteobacteria)，γ-变形菌亚门(γ-Proteobacteria)、疣微菌门(Verrucomicrobia)和酸杆菌门(Acidobacteria)。(3)不同生境16SrRNA基因的比较分析表明，东湖沉积物细菌群落与多种生境II华中科技大学硕士学位论文微生物具有相似性，与富营养化东平湖和洪湖昀为相近。2.关于NirS类反硝化细菌多样性及系统发育分析(1)物种多样性统计表明，在T样点，反硝化细菌多样性指数与丰富度指数昀高；相反，M样点反硝化细菌物种多样性及丰富度指数较低。结合环境因子结果分析表明，东湖沉积物中TN、NH4+和NO3-的浓度可能是影响NirS类反硝化细菌多样性和空间分布的重要因素之一。(2)nirS基因多样性及系统发育分析表明，反硝化细菌具有丰富的系统发育多样性。东湖富营养沉积物生境NirS类反硝化细菌有三个显著分群。统计表明，群I占总群体的67.7%，广泛分布于不同的生态环境；来自郭郑湖代表菌的81%分布于群I，而庙湖的代表菌中65%分布于群II。(3)相似性环境分析表明，东湖沉积物反硝化细菌类群与人工湿地生境反硝化类群具有较高相似性。两种生境序列间分析表明，群I及群II微生物是富营养化生境沉积物NirS类反硝细菌的优势类群，在反硝化过程中可能扮演重要角色。关键词：16SrRNA基因，亚硝酸盐还原酶基因，反硝化作用，限制性片段长度多态性III华中科技大学硕士学位论文AbstractAquaticeutrophicationhasbecomeoneofthemajorfactors,whichseriouslyimpactsontheecologicalsecurity,economicandsocialdevelopmentofChina.Microbesasthedecomposersinwaterecosystemsplayanimportantroleinthemetabolismofnitrogenandphosphorusnutrients.However,studiesonthecommunitiystructureanddiversityofmicrobialineutrophicwatersbyusingmolecularecologicaltechnologyarestillfewinChina.Moreover,thestudiesonspecificfunctionalgroupsofmicrobeswereevenfewer.Therefore,itisveryimportanttostudythediversity,distributionandfunctionofthemicrobes,especialthefunctionalmicrobialcommunityintheeutrophicwaters.EastLakeinWuhanCity,Chinaisatypicaleutrophiclake.Thesedimentsampleswerecollectedbyfive-pointsamplingmethodfromitsfourtypicalsub-lake,GuozhengLake,TanglingLake,TuanLakeandMiaoLakeinSeptember,2009.Basedonclonelibraries,restrictionfragmentlengthpolymorphism(RFLP)methodandsequencinganalysis,bacteriacommunitystructureanddiversitywereinvestigated.Moreover,thephylogenydiversityofthenirS-denitrifierscommunitiesinthesedimentswasalsostudied.Themainresultsarelistedasfollows:1.BacterialdiversityandphylogenyinthesedimentsoftheeutrophicEastLake.⑴EnvironmentalparametersshowedthatthesedimentfromMiaoLake(Msite)containedthehighestamountoftotalnitrogen(TN)andNH4+-N,whilethatfromTuanLake(Tsite)containedthelowest.Thediversity,richnessandevennessstatisticscalculatedbyDOTURsoftwareindicatedthatamongthefoursub-lakes,TuanLakeharbouredthehighestdiversityandrichness,whileMiaoLakehadthelowest.TheresultssuggestedthatthediversityindicesofthebacteriacommunitywereprobablyaffectedbythecontentofTNandNH4+nutrientsinthesediments.⑵NJtreeconstructedbyusingthePHYLIPsoftwareshowedthatahighdiversityofmicrobespeciesinhabitedintheDonghusediments.PylogeneticanalysisdeterminedatthephlumlevelhasdividedthemintothephylaofProteobacteria,Acidobacteria,Bacteroidetes,Chloroflexi,Firmicutes,Verrucomicrobia,Planctomycetes,CrenarchaeotaandEuryarchaeota.PhylalikeProteobacteria,Acidobacteria,Bacteroidetes,Chloroflexi,IV华中科技大学硕士学位论文FirmicutesandVerrucomicrobiawerepresentinall4samplingsites.However,PlanctomyceteswasabsentfromsiteT,andArchaea(CrenarchaeotaandEuryarchaeota)wereonlyfoundinsedimentsamplesofMsite.Statisticsindicatedthatδ-Proteobacteria,β-Proteobacteria,γ-Proteobacteria,AcidobacteriaandVerrucomicrobiawerethedominantmicroorganisms.⑶BLASTresultssugguestedthatthemicrobialcommunityinthesedimentsofEastLakewassimilartothosefromvarioushabitats,especiallyfromtheeutrophicDongpingLakeandHonghuLakeinChina.2.GeneticdiverisyandphylogenyofnirS-denitrifiersintheeutrophiedDonghuLake⑴Thediversityandrichnessindicesshowedthatamongthefoursub-lakes,TuanLake(Tsite)harbouredthehighestdiversityandrichnessofNirS-likedenitrifiers,whilethedenitrifiersinMiaoLake(Msite)wasthelowest.Coupledwiththeenvironmentalfactorsdetermined,itwassuggestedthatthediversityindiceswasaffectedbythecontentofTN,NH4+andNO3-nutrientsinthesediments.⑵PhylogenicanalysissuggestedthatsedimentaryNirS-likedenitrifiersintheEastLakecouldbeclassifiedintothreegroups,GroupI,II,III.GroupIaccountedfor67.7%ofalltestedcommunities.81%sequencesfromGuozhengLakewereclusteredintoGroupI,while67.7%sequencesfromMiaoLakewereclusteredintoGroupII.⑶ComparativeanalysisofcommunitiesfromEastLakeandartificialwetlandfoundthattheywerecloselyphylogeneticallyrelated.ItindicatedthatGroupIandIImightplayanimportantroleinthedenitrificationintheeutrophiedlakes.Keywords:16SrRNAgene,nirSgene,Denitr