不同处理油松cdna-srap pcr体系建立及基因表达差异分析

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1、EstablishmentofcDNA-SRAPPCRReactionSystemandGeneExpressionDifferenceAnalysisofChinesePinewithDifferentTreatmentsCandidate：LiuYangSupervisor．Prof．GaoBaojiaMajor：．EcologyAbstractInthisstudy,Chinesepine俨切淞tabulaeformisCarr)forestsindifferentstandtypesinPingquanCountyofChengdeCity,Hebeiprovin

2、cewastakenaStheresearchobject，bycombiningtheanalysisoffieldtestandmolecularexperiment．Wesetthecontroltreatment，treatmentofChinesepinefedby10pinecaterpillars(Dendrolimustabulaeformis)perplant，treatmentofChinesepinefedby30pinecaterpillarsperplantandtreatmentofcuttingleavies(thedamagedegreee

3、qualstothetreatmentofChinesepinefedby30pinecaterpillarsperplant)．CombinatingofeDNASRAPmethodsforgeneexpressiondifferenceanalysismethod，wepreliminarystudiedthegeneexpressiondifferenceofChinesepineunderdifferentstandtypesanddifferenttreatments，analyzedthereasonofthedifferences，predictedthef

4、unctionofthegeneandprovidedtheevidenceofgenelevelfortheinducedresistancemechanismwhichgeneratedbytherelationshipbewteenChinesepineandpinecaterpillarundertheinteractionofforesttypesandChinesepinecaterpillarherbivory．111emainresultsareasfollows：(1)ThesystemofPCRwasoptimizedinfourleveloffive

5、factors，whichWasTaqDNApolymerase，Template，primer,M92+anddNTPbyusingorthogonaldesignL16(45)．Theresults，generatedbySRAP-PCRwereanalyzedbysoftwareSPSS，showedthattheeffectsofthefivefactorsontheamplificationsystemdecreasedinthefollowingorder：dNTP>cDNAtemplate>TaqDNApolymerase>primer>M92+．Asuit

6、ableSRAP-PCRsystemforChinesepinethatiscontaining2．0UTaqDNApolymerase，80～120ngcDNAtemplate,0．4I．tmol／Lprimer,1．5mmol／LM92+，0．2mmol／LdNTPintotal20oLreactionsystem．ThisoptimizedsystemforChinesepinewouldbeusefulindifferentialgeneexpressionanalysisbyusingthecDNA—SRAPtechnique．Reaction：Protocol

7、Wasinitiallydenaturedat94"Cfor5min；denatuered94℃f10r50S，annealedat35"Cfor50S，extensedat72℃forlmin，5cycles；denatueredat94℃for50S，annealedat50℃for50S，extensedat72℃forlmin，35cycles；extensedat72℃for7min；storedat4"C(2)UsingtheoptimizedsystemofcDNA-SRAP,225pairsofSRAPprim