分子生态学技术

分子生态学技术介绍种群多样性和动态种群丰富度结构和功能的关系MethyleneBlueStainNon-PremoversPolyphosphateAccumulatingOrganisms(PAOs)Thetop-viewCSLMmicrographsof60-day-oldanodebiofilmformedbymixedcultureinsingle-chamberair-chatodeMFCfedwithacetate.Livecells(syto9)eDNA(DDAO)Exopolysaccharides(AlexaFluor633conjugate,concanavalinA)微生物生态系统的分子检测技术收集样品核酸提取和电泳rRNAgene克隆rRNAgenes分类与发育分析探针技术如荧光原位杂交技术(FISH)6TypeSizeLargesubunitSmallsubunitprokaryotic70S50S(5S,23S)30S(16S)eukaryotic80S60S(5S,5.8S,28S)40S(18S)Bacterial16S,23S,and5SrRNAgenesaretypicallyorganizedasaco-transcribedoperon.Theremaybeoneormorecopiesoftheoperondispersedinthegenome(forexample,Escherichiacolihasseven).ArchaeacontainseitherasinglerDNAoperonormultiplecopiesoftheoperon.The3'endofthe16SrRNA(inaribosome)bindstoasequenceonthe5'endofmRNAcalledtheShine-Dalgarnosequence.smallsubunitribosomalRNA(SSUrRNA)79在分子生态学中，生物的核糖体RNA基因（rDNA）由于其自身的特征，已经成为最重要的靶基因，各种方法的实现都是通过对rDNA的操作来完成的探针(probe)杂交为基础的分子生物学技术：FluorescenceinsituHybridization，FISH;MicroarrayPCR为基础的基因指纹(GeneticFingerprintingProfiles)技术：DenaturingGradientGelElectrophoresis,DGGETerminalRestrictionFragmentLengthPolymorphism,T-RFLP测序技术（sequencing）非培养技术1以探针杂交为基础的技术：FISH原理：是以荧光素标记的特异寡核苷酸为探针，根据碱基互补配对的原理，同微生物细胞内含有互补序列的基因组特异性地杂交，再通过荧光显微镜等检测杂交的细胞。Ref:DeLongetal.Phylogeneticstains:ribosomalRNA-basedprobesfortheidentificationofsinglemicrobialcells.Science.1989,65:5554-5563☆-TAGCAGTTCATCGTCAAG||||||||||||||||||||||☆-TAGCAGTTC实现步骤：各种FISH不同之处：探针序列，杂交条件和漂洗条件。对活性污泥或纯培养微生物进行FISH的主要步骤如下图所示:细胞预处理及固定固定细胞处理及杂交漂洗，去除未杂交探针荧光显微镜检测部分荧光色素的激发波长和发射波长荧光色素激发波长/nm发射波长/nm颜色AMCA350450蓝色PE(phycoerythrin)450～470575橙色FITC490～495515～517黄绿色6-FAM√494518绿色TET521538橙色HEX√535553粉红色Cyanins3√554568～574橙色TAMRA560582玫瑰红TRITC575600深红色ROX587607红色Texasred595615红色Cyanins5684665红色FISH杂交中应用的寡核苷酸探针探针序列(5’3’)特异性靶位点ARCH915GTGCTCCCCCGCCAATTCCTArchaea16SrRNA,915-934EUB338GCTGCCTCCCGTAGGAGTEubacteria16SrRNA,338-355NHGCTATAGTTACGGCCGCCGTLow%G+CBacteria23SrRNA,1901-1918HGC69aTATAGTTACCACCGCCGTHigh%G+Cgram-positivebacteria23SrRNA,1901-1918ALF1bCGTTCG(CT)TCTGAGCCAGα-Proteobacteria16SrRNA,19-35BET42aGCCTTCCCACTTCGTTTβ-Proteobacteria23SrRNA,1027-1043GAM42aGCCTTCCCACATCGTTTγ-Proteobacteria23SrRNA,1027-1043SRB385CGGCGTCGCTGCGTCAGGδ-Proteobacteria,somegram-positives16SrRNA,385-402DSV698GTTCCTCCAGATATCTACGGDesulfovibrionaceae16SrRNA,698-717InsituhybridizationofactivatedsludgefromthedeterioratedEBPRreactor.Theleftsideshowsphase-contrastimages,andtherightsideshowsepifluorescencemicrographsofthecorrespondingareas.(A)InsituhybridizationwithprobesspecificforthedomainBacteria(green)andthegammasubclassoftheProteobacteria(red).(B)InsituhybridizationwithprobesspecificforthedomainBacteria(green)andforthenovelsubgroupofthegammasubclassoftheProteobacteria;theprobespecificforthebacteriafromband4isshowninblue,andtheprobespecificforthebacteriafrombands3,5,and6isshowninyellow.Thescalebarappliestoalloftheimages.Ref:Nielsenetal.Appl.Environ.Microbiol.,1999,65(3):1251-1258Bacteria(green)GammaProteobacteria(red).Bacteria(green)GammaProteobacteria(red).Band4GammaProteobacteria(red).Band3,5Example,thestartlingdiscoverofdenseclusteringofnitrifyingbacteriausingFISHAmmoniaoxidizersinlargeredclusterNitriteoxidizersinSmallgreenclustersAllwithinamuchlargerbiomassaggregateofheterotrophs,inertbiomass,andEPS(leftpanel).GenechipGeochipCommunityGenomeArraysWholeGenomeMicroarraysFunctionalGeneArraysGeneExpressionPatternsProteinarrayMicrobialCommunityDiversity&MechanismsGenomicTechnologyMicrobialfunctionalGenomicsMicrobialEcology&ExtremophilesOligonucleotideArraysProducingMagneticNanoparticlesUraniumReductionCommunity&EcosystemGenomicsGenechipGeochip2基因指纹技术:DGGE,T-RLFP2.1.DGGE/TGGE原理：根据序列、组成不同的双链DNA解链温度不同，从而在变性梯度胶中解链的时间不同（一旦解链，泳动速度急剧下降）而将它们分开。Ref:Muyzeretal.Profilingofcomplexmicrobialpopulationsbydenaturinggradientgelelectrophoresisanalysisofpolymerasechainreaction-amplifiedgenescodingfor16SrRNA.Appl.Environ.Microbiol.1993.59:695-700.加热或加变性剂提高温度或变性剂浓度高温解链区低温解链区双链DNA解链温度实现步骤：A.一个引物的5‘端需要有40BP左右的GC-CLAMP，PCR扩增。B.制备梯度胶，凝胶混合器。C.电泳及染色。E.harbinenseA.elongatumM.cerevisiaeClostridumsp.E.harbinenseA.elongatumM.cerevisiaeE.harbinenseC.pasteurianumClostridumsp.E.harbinensePropionicimonassp.A.elongatumClostridumsp.DGGEprofilesofV3regionof16SrRNAgeneinthemicrobialcommunitiesfromthreereactors系统发育树2.2T-RLFP原理：采用限制性内切酶将PCR扩增（在一个引物或两个引物的5‘采用荧光素标记）的产物进行酶切，跑测序胶，结果直接采用自动测序仪进行扫描。实际是将带型图谱转化为峰值图谱Ref:Liuetal,Appl.Environ.Microbiol.,1997,63(11):4516-452225Electropherogramsofthe5’(A)and3’(B)T-RFLPsofHhaIdigested16SrRNAsamplifiedfromasix-memberbacterialcommunitycorrespondingtoHhaIdigestion.实现步骤：a.采用荧光标记的引物进行PCR扩增；b.产物酶切；c.制胶；d.电泳及峰值图谱的获得3.焦磷酸测序(Pyrosequencing)技术28元基因组学(Metagenomics)