cailei_真菌分子系统学

真菌分子系统学蔡磊中国科学院微生物研究所真菌学国家重点实验室Email:[e-mail]分子系统学原理和方法系统学(Systematics)研究生物的分类与命名的科学。附属学科：分类学taxonomy命名法nomenclature系统演化(发育)学(Phylogenetics)研究生物间的亲缘关系及其演化的科学。在真菌中，传统上以比较形态学、细胞学、超微结构和细胞壁化学组分等为依据，在很小程度上也以化石为依据,现在主要依靠DNA序列分析为依据分子系统学(MolecularSystematics,MolecularPhylogenetics)用遗传标记或遗传物质(geneticmarkersorgenemarkers)为依据在分子或基因水平上进行生物系统演化的研究。TotalDNAhybridizationRFLPsanonymouscloneRAPDsmtDNArDNAMappinganonymousclonemtDNA(wholemolecular)rDNAspacersrDNAgenes(mtornuc)SequencingSpacers(ITS)mtrDNAgenesnucrDNAgenesProteingenesModeratelyconservedconservedPopulationgeneticsPopulationspeciesGeneraFamilies&OrdersClasses&DivisionsKingdomPhylogeneticrelationships真菌分子系统学研究常用方法及其适用范围(Brunsetal.,1991)检测两个菌株的DNA变性后形成的单链DNA进行杂交时复性的程度。两个菌株的变性DNA混合在一起，在一定条件下单链DNA分子复性形成杂合分子。两个菌株的亲缘关系愈近，它们的DNA序列愈相似，可以形成更多双链DNA分子。比较杂合DNA分子间和对照同源DNA(homoduplexDNA)间复性时形成复性分子的差异，可确定两个菌株间的DNA相似性(DNA-DNAsimilarity;DNA-DNArelatedness;DNA-DNAhomology)测定方法固相法：测相对结合率(relativebindingratio,RBR)液相法，测熔点温度差异(ΔTm).DNA-DNA杂交DNA-DNA杂交实验原理DNA-DNA杂交应用：在真菌中多用于酵母菌的分类(近缘种间的鉴别)菌株间DNA-DNA相似性70%，同一个种，40%～60%，变种或根据不同的情况而定；40%，不同种。RFLP(RestrictionFragmentLengthPolymorphisms)1.细胞总DNA经限制性内切酶消化后进行琼脂糖凝胶电泳，产生由不同大小的DNA片断形成的一条连续的电泳带，将DNA转移到特定膜上(SouthernBlotting)，再用标记的DNA探针进行杂交，产生可分辨的杂交带。2.原理:该技术是利用限制性内切酶能识别DNA分子的特异序列，并在特定序列处切开DNA分子，即产生限制性片段的特性，对于不同种群的生物个体而言，他们的DNA序列存在差别。如果这种差别刚好发生在内切酶的酶切位点，就导致了用限制性内切酶酶切该DNA序列时，就会少一个或多一个酶切位点，结果产生少一个或多一个的酶切片段。这样就形成了用同一种限制性内切酶切割不同物种DNA序列时，产生不同长度大小、不同数量的限制性酶切片段。后将这些片段电泳、转膜、变性，与标记过的探针进行杂交，洗膜，即可分析其多态性结果RFLP分析RFLPpatternsobtainedafterdigestionofnuclearDNAofAspergillusspecieswithKpnIandXhoIandprobingwiththe0.9-kb28SrRNAEcoRIfragmentofAgaricusbisporus(Pařenicovaetal.,2001).RAPD(RandomAmplifiedPolymorphicDNA)：用短的任意序列的寡核苷酸片断作为引物，对模板DNA或RNA进行PCR扩增，产生大小不同的随机产物，用普通电泳进行多态性显示和比较。应用：近似种间或种内菌株和群体间遗传差异的比较。RAPD分析RAPDfingerprintingofthethreespeciesinthePenicilliumroquefortigroupusingeitherNS2(a)orNS7(b)asaprimer(Boysenetal.,2000).脉冲电泳(Pulsed-FieldGelElectrophoresis,PFGE)的原理及优势原理：电场不断改变,迫使DNA分子的泳动方向做相应的改变。小的DNA分子比大的变形快，故前者比后者泳动速度快，从而将不同大小的DNA分子分开。优势：分辨率可达10,000Kb(10Mb)以上E1E2用脉冲电泳核型分析估测的部分植物病原真菌基因组大小和染色体条数(Walz,1995)。种类染色体数Cladosporiumfulvum9Cochliobolusheterostrophus15-16ColletotrichumgloeosporioidestypeA13-15tpyeB6-8Erysiphegraminis7Fusariumoxysporumf.sp.conglutinans8Fusariumoxysporumf.sp.raphani11Leptosphaeriamaculanshi.viwe.vi6-812-14Magnaporthegrisea6-10Nectriahaematococca10-20Ophiostomaulmi6Phomatracheiphila12Phytophthoramegasperma10-13Septorianodorum14-19Septoriatritici17-18Tilletiacaries12-15Tilletiacontroversa12-16Ustilagohordei16-21Ustilagomaydis20Ustilagotritici13用脉冲电泳核型分析估测的部分非病原真菌基因组大小和染色体条数(Walz,1995)。种类基因组(Mb)染色体条数Absidiaglauca429-10Acremoniumchrysogenum328Acremoniumflavum26.57Acremoniumstrictum27.77Agaricusbisporus13Aspergillusnidulans318Aspergillusniger35.5-38.58Aspergillusoryzae8Cephalosporiumacremonium22.58Curvularialunata29.712Histoplasmacapsulatum7Neurosporacrassa477Penicilliumjanthinelum39-468-10Phanerochaetechrysosporium3211Podosporaanserina33.47Schizophyllumcommune35-366Tolypocladiuminflatum30.57Trichodermalongibrachiatum337Trichodermareesei386DNA(或RNA)特定区段的碱基序列测定和比较18S5.8SITS226SITS1D1/D2DNASequencingWhatisPhylogenetics?PhylogenyreflectstheprocessofevolutionUsestreediagramstoportrayrelationshipsbaseduponrecencyofcommonancestryHowarephylogeniesinferred(estimated)?phylogeniescanonlybeinferred,neverknowntraditionally,phylogenetichypotheseswerederivedbyintuition(直觉),perhapsguidedbyideasregardingthelawsofevolution,inmodernsystematics,phylogenetichypothesesarecreatedthroughnumericalanalyses(phylogeneticanalyses)ofcharactersEVOLUTIONCharlesDarwinTheOriginofSpecies(1859)organismsdescendwithmodificationfromcommonancestors;variationsareheritableinanyspecies.CLASSIFICATIONBASEDONSIMILARITYCarolusLinnaeus(1707-1778)GrouporganismsaccordingtophenotypicresemblanceCLASSIFICATIONBASEDONGENEALOGYWilliHennig1966.PhylogeneticSystematics.UniversityofIllinoisPress.StudyofSISTER-GROUPRELATIONSHIPSTRADITIONALAPPROACHThreeextantspecies,A,BandCCBATRADITIONALAPPROACHFossiltaxonXfound,withgreatersimilaritytoAandBCBAXTRADITIONALAPPROACHCBAXFossiltaxonYfound,withgreatersimilaritytoXandCYHENNIG’SAPPROACH:CLADISTICSWILLIHENNIG(1913-76)proposedtheuseofSISTER-GROUPRELATIONSHIPSSISTER-GROUPRELATIONSHIPSThreeextanttaxa,A,BandCSISTER-GROUPRELATIONSHIPSAandBarebelievedtobemorecloselyrelatedtoeachotherthaneitheristoCSISTER-GROUPRELATIONSHIPSOnthisbasis,relationshipscanbededucedWhatkindsofcharactersareusedinphylogeneticanalysis?-physicalform(morphology,anatomy),-biochemicalcharacters,-behavioralcharacters…geneticbasisofthesekindsofcharactersisnotalwaysknownmanyphylogeneticcharactersarenowderivedfromDNAsequence.DNAsequencesarenowthepredominantsourceofphylogeneticcharacters,butthereareotherkindsofmolecularcharactersAremolecularcharacterssuperiortomorphologicalcharactersforphylogeneticinference?yesandno….strengthsofmolecularcharacters:verynumerousallowexplicitcoding(A/C/G/T)canbecomparedinorganismswithnomorphologicalsimilaritiesmodelsofevolutionbasedongeneticcod