基因表现连续分析法(SAGE)及其在植物研究之应用农业试验所王强生廖

1SAGE(cloningbydifference)RNART-PCR(subtractivehybridization)EST(comparativeESTanalysis)(profiles)(differentialscreening)(differentialdisplay,DD)mRNAVelculescu(1995)(serialanalysisofgeneexpression,SAGE)SAGE(post-genomicera)SAGENlaIIIcDNA3’9-11DNA()Linker-LigationsPCRmRNA(abundance)9(49)15,000–33,000SAGE2SAGE()TotalRNAmRNA1.phenol/chloroform()totalRNASAGEtotalRNA1mg(mRNA5%)2.Dynal®Co.Dynabeads®Oligo(dT)25polyARNA5gpolyARNAcDNA3.firststrandedbufferdNTPmixturebiotinylatedoligodTprimer5gpolyARNA6510RNA3MMLVreversetranscriptase37cDNA4.3cDNA:RNARNaseHDNApolymeraseIcDNAcDNA1.cDNA(CATG)(frequentcutenzyme,NlaIII)(AE)cDNAstreptavidinbeadspolyA3'cDNA2.cDNALinkerALinkerB(linkerTypeIIS)TypeIIS(FokI)(TE)3.cDNAKlenowfragment(fillin)(bluntSAGESAGE)phenol/chloroformcDNAligation4.cDNAphenol/chloroform(NlaIII)cDNA(ditag)26-30(bases)5.cDNAcDNA(sizefractionation)2006.()SAGEBLASTanalysisSAGEa.(match)b.ESTs(expressedsequencetags)mRNAc.mRNASAGE(transcriptprofiling)p53(apoptosis)SAGEMatsumura(1999)SAGESAGESAGEMatsumura(1999)(profile)RNAcDNASAGE10,1225,5935,921SAGE()10080%cDNASAGEDNASAGE(housekeepinggenes)SAGE(anaerobictreatment)SAGE2,0941,4962,2051,551()2486SAGE(10%)(20%)()()SAGESAGERACE(rapidamplifiedcDNAend)9-13SAGE1005(M13forwardT7promoter)DNAPCRcDNA9-13SAGE1.2.3.()()SAGEmRNA(2.5-5µg)SAGEmRNAMicroSAGE(Datsonetal.,1999)SAGEPCR(ditag)MicroSAGESAGESAGE1.Datson,N.A.,vanderPerk-deJong,J.,vanderBerg,M.P.,deKloet,E.R.,andVreugdenhil,E.1999.MicroSAGE:amodifiedprocedureforserialanalysisofgeneexpressioninlimitedamountsoftissue.Nucl.Acid.Res.27:1300-1307.2.Matsumura,H.,Nirasawa,S.,andTerauchi,R.1999.Transcriptprofilinginrice(OryzasativaL.)seedlingsusingserialanalysisofgeneexpression(SAGE).PlantJ.20:719-726.3.Velculescu,V.E.,Zhang,L.,Vogelstein,B.,andKinzler,K.W.1995.Serial6analysisofgeneexpression.Science270:484-487.4.Zhang,L.,Zhou,W.,Velculescu,V.E.,Kern,S.E.,Hruban,R.H.,Hamilton,S.R.,Volgelstein,Kinzler,K.W.1997.Geneexpressionprofilesinnormalandcancercells.Science276:1268-1272.5.SAGE(GMO)PCRSAGERAGE(rapidanalysisofgeneexpression)