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mRNA1,2,2,2*(1.,410128;2.,410125)mRNA,,,mRNA,PCRdNTP7mRNAmRNA;;Q789A0517-6611(2007)13-03820-02ApplicationofmRNADifferentialDisplayTechniqueinIsolationofResistantGenesinRiceCHENHaoetal(LaboratoryofCellEngineering,HunanAgriculturalUniversity,Changsha,Hunan410128)AbstractThetechniqueofmRNAdifferentialdisplayhasextensivelybeenusedinisolationofresistantgenesinricesinceitsnaissance.Throughtheanalysisof1thedifferentiallyexpressedgenes,theresistantmechanismofricecouldbefurtherclarified.TherecentstudiesonisolatingresistantgenesinricebymRNAdifferentialdisplaytechniqueweresummarizedandtheimprovementsofDDRT-PCRwasexpoundedsimplyfrom7aspectsofimprovementofprimer,PCRannealtemperature,improvementofdNTPconcentration,selectionofglueconcentration,improvementofmarkermethodandimprovementofdifferencesegmentvalidateKeywordsmRNAdifferentialdisplay;OryzasativaL.;Resistantgene,15%mRNA1Liang1992mRNA(mRNADifferentialDisplay,DD)(ReverseTranscription,RT)(PolymeraseChainReaction,PCR)(PAGE)[1]mRNA3poly(A),oligo(dT)12MN(MACG,NACGT),12,RNAcDNA,mRNA12mRNA-cD-NA,512PCR,,50100100500bpDNA,mRNA,cDNA22.1,,77-170M-20,13200600bpcDNA[2]mRNApp2462-11Pedel,,,25(KZCX3-SW-434);(03JJY1004);(1982-),,,,:*2007-02-02,4,,[3]LimRNA1A(REF1A),Northern1AABA[4],1A,1A,IPT(Isoproth-iolane)46,IPT,IPT,mRNA5[5]Watanabe,mRNAcDNA,ETR2EIN450%52%,cDNAOs-ERL1,Os-ERL1[6]TyagiN22,cDNA,Northern,DIpsbA[7]Zhou,OsMT(),,OsMT2,OsMT[8]LiumRNA,90kDa(rHsp90),pH[9],JournalofAnhuiAgri.Sci.2007,35(13):3820-3821,38452.2mRNADong,,IR2630,mRNA,NorthernIR26[10]mRNA,cDNA,463bp,poly(A)AATAAA,poly(A),[11]QinmRNA,131(Aichi-asahi)8cDNA,321bp,cDNA,12[12],cDNAcDNAcDNA96%,,2Southern,2Northern,,ZhangmRNA,87,816,11RPR1,RPR1[13]CBB23JG30,P6mRNA,NorthernCU10,,,,CU10[14]33.1mRNAoligo(dT)12MN,,LiangT11M[15],,T11MNmRNA3.2PCR10,PCRPCR42,;40,,4042[16]3.3dNTPDDRT-PCRdNTP,,2mol/LdNTP,[17]3.4DDRT-PCR6%(PAGE)AverboukhColleaguesGenomyxLRTM,1.5kb[18]3.5mRNA,,,[19][20][21][22]3.6NorthernNorthernmRNA,Northern[23]PCR[24]3.7,PCRPCR[25],,,,[25]4mRNA,,,mRNA[1]LIANGP,PARDEEAB.DifferentialdisplayofeukaryoticmessengerRNAbymeansofthepolymerasechainreaction[J].Science,1992,257(5072):967-971.[2],.DDRT-PCR[J].,1995,25(8):840-847.[3],,,.DD-PCR[J].,1997,30(5):71-74.[4]LIZY,CHENSY.Inducibleexpressionoftranslationelongationfactor1Ageneinriceseedlingsinresponsetoenvironmentalstresses[J].ActaBotanicaSinica,1999,41(8):800-806.[5],,,.IPTmRNA[J].,2001,27(6):935-940.[6]WATANABEH,SAIGUSAM,HASES,etal.CloningofcDNAencodinganETR2-likeprotein(Os-ERL1)fromdeepwaterrice(OryzasativaL.)andin-creaseinitsmRNAlevelbysubmergence,ethylene,andgibberellintreatments[J].JournalofExperimentalBotany,2004,55(399):1145-1148.[7]TYAGIA,CHANDRAA.IsolationofstressresponsivePsbAgenefromrice(OryzasativaL.)usingdifferentialdisplay[J].IndiaJBiochemBiophys,2006,43(4):244-246.[8]ZHOUG,XUY,LIJ,etal.Molecularanalysesofthemetallothioneingene-familyinrice(OryzasativaL.)[J].JBiochemMolBiol,2006,39(5):595-606.[9]LIUD,ZHANGX,CHENGY,etal.rHsp90geneexpressioninresponsetoseveralenvironmentalstressesinrice(OryzasativaL.)[J].PlantPhysiolBiochem,2006,44(5/6):380-386.[10]DONGHT,WUYL,CHENGZQ,etal.Molecularcloningofdifferentiallyexpressedgenewithkinasemotifinducedbyxanthomonasoryzaepv.oryzae[J].JournalofZhejiangAgriculturalUniversity,1998,24(5):548-552.[11],,,.mRNAcDNA[J].,2000,16(2):124-126.[12]QINQM,ZHANGQ,ZHAOWS,etal.Identificationofalectingenein-ducedinriceinresponsetomagnaporthegriseainfection[J].ActaBotanica(3845)12833513mRNA2.22,,,,,,,2,,2000,,B;2001,2000;20022000,,B2%mm20000.800.42A,B,C,D18.600.2820012.000.37A,B,C,D65.800.2420020.770.40A,B,C,D18.200.272.33,A,B,CD20002002C;2001BC,ACDC,;20002002,2.4(4),3(1),,(2),,(3),710d32000%%Vcmg/100g2001%%Vcmg/100g2002%%Vcmg/100gA17.32.9016.2016.33.146.0217.52.7117.80B18.12.9619.5017.03.209.4117.82.8518.90C17.52.9116.5016.43.176.2418.22.9016.70D17.82.9217.8016.83.146.3617.22.7919.1018.73.148.3817.83.356.7718.63.088.474gkg/cm245dgkg/cm290dgkg/cm28565.858485.448234.948255.408165.017864.18(4),[1].[M].:,2003.[2],,,.[J].,2002(4):12.[3],,,.[J].,2003,23(3):255-259.[4],,.[M].:,1999:9.[5],,,.[J].,1998(9):43.[6].[J].,1998(2):13-14.(3821)Sinica,2003,45(1):76-81.[13]ZHANGHY,LIUY,LIUDC,etal.Identificationofgenesrelatedtoresis-tancetoMagnaporthegriseausingdifferentialdisplaytechniqueinrice[J].YiChuanXueBao,2005,32(7):719-725.[14],,,.mRNA[J].,2005,31(10):1373-1376.[15]LIANGP,ZHUW,ZHANGX,etal.Differentialdisplayusingone-basean-choredoligodTprimer[J].NucleicAcidsResearch,1994,22:5763-5764.[16],.[M].:,2002:215.[17],,,.[J].,2003,19(1):55-59.[18]AVERBOUKHL,DOUGLASSA,ZHAOS,etal.BettergelresolutionandlongercDNAincreasetheprecisionofdifferentialdisplay[J].BioTechniques,1996,20:918-921.[19]TAKASHII,KEIJIK,NAOKIA,etal.Fluorescentdifferentialdisplay:arbi-trarilyprimedRT-PCRfingerprintingonanautomatedDNAsequencer[J].FEBSLetters,1994,351(2):231-236.[20].[D].:,2006.[21]GANGA,LUOGZ,VELTRIRW,etal.Sensitive,nonradioactivedifferen-tialdisplaymethodusingchemiluminescentdetection[J].BioTechniques,1996,20(3):342-346.[22]JEREMYJW,CHENJJ,PECKK.Non-radioisotopicdifferentialdisp