烟草腺毛全长cDNA文库的构建

456()Vol.45No.6200611JournalofXiamenUniversity(NaturalScience)Nov.2006cDNA1,1,1,23(1.,,450002;2.,361005):2006205219:(110200101005):(1966),,,.3:[e-mail]:K326,TrizolRNA,SMARTcDNA.,1.01106pfu/mL,99.5%.24,,8001000bp,Unigene,23Unigene,21,14,66.7%.,,.:;;cDNA:Q785:A:043820479(2006)0620859204,.,[1],,,.,,,,,.[2].,,[3].,.,,.Croteau[4]EST,48%,252(dxr),,50%,.EST,,.KentuckyWagnerTI1068cDNA,P450,[5];,[6].,cDNA,,.11.1K326.Erming[7].,,,,,.5min,,,,.1.2RNAGIBCOBRLTrizolreagent.0.56g,,,5.5mLTr2izol;,,75%,Milli2Q,.1.3cDNA3L,,cDNA.LDPCRcDNA,,95HYBAIDPCREX2PRESSPCR,9515s,686min,30.5L1.1%.1.4cDNA50LdscDNA(23g)0.5mL,K,::,SfiI,CHROMASPIN2400,cDNA10,1.1%cDNA.1.5cDNApBluescriptSKpBluescriptIISK,EcoRINotISfiIASfiIB.,cDNA(SfiIASfiIB).,cDNA3:1.1.6200L(DH25)50mL,,1L,45min,4290s,2mLLB(),37,150r/min,45min,3000r/min10min,250LLB.1.7200L15cm(Apr2IPTG/x2galLB),37,,[8]:=1000L/mL(L).245,cDNA,24Unigene,..2,.1,,,1Fig.1ThetrichomeofNicotinantabacium,,.,,,RNA.,5min,,.TrizolRNA,RNA0.95g,,OD260/OD280=1.924,RNADNA.DEPCRNA,0.095g/L.RNA,2,12RNA70460min1.1%,:23sRNA18sR2NA,23sRNA18sRNA,RNA,[9].LD2PCRcDNA,1.1%,,1600bp,PCRcDNA,cDNA(3).2RNA1.70;2.4Fig.2ElectrophoresisoftotalRNAcDNASfiI,SfiI,Chromaspin2400,8.1.1%,068()20063cDNA1.cDNA;M.MarkerFig.3ElectrophoresisofdscDNAcDNAcDNA,1600bp,,cD2NA(4).48,cDNA.3:1pBluescriptIISK.(DH25),Apr2IPTG/x2galLB,.1012/;,5/,99.5%.pBluescriptIISK,SfiI,.1.01106pfu/mL,20L,-80.4cDNASfiI,CHRO2MASPIN240018.8;M.Gibco/BRL1kbDNAladder(122161119810180916281447126610850904072305420361636101850639634429822020115413475bp)Fig.4ElectrophoresisofdscDNAofSfiIdigestionandcDNAsizefractionationbyCHROMASPIN2400,245’,Unigene.24,23Unigene,Uni2gene95.8%.,2421,14,:14/21100%=66.7%.cDNA.3,cDNA,.,,cDNA[9].,,TrizolRNA,SMART2cDNA.SMARTcDNAmRNA5SMARTIII,LDPCRcDNA,cDNA,SMARTIII.cDNApBluescriptIISK,SfiIASfiIB,cDNA,DH25,cDNA.1.01106cfu/mL,99.5%,800bp,66.7%,.cDNA,.,,,,,.:[1],.[M].:,1998.[2],.[J].,1993,4:32-36.[3]WagnerG.Leafsurfacechemistry[M]PPDavisL,NielsenT.Tobacco:Production,ChemistryandTechnology.Ox2ford:BlackwellScienceLimited,1999.[4]LangeM,CroteauR.IsoprenoidbiosynthesisviaaMeval2onatePathwayinplant:cloningandheterologousexpressof12deoxy2D2xylulose252phosphatereductoisomerasefromPeppermint[J].ArchivesofBiochemistryandBiophysics,1686:cDNA1999,365:170-174.[5]ErmingW,JosephDP.SuppressionofP450hydtoxylasegeneinplanttrichomeglandsenhancesnatural2product2basedaphidresistance[J].NatureBiotechnology,2001,19:371-374.[6]MahmoudS,CroteauR.Metabolicengineeringofessentialoilyieldandcompositioninmintbyalteringexpressionofdeoxyxylulosephosphatereductoisomeraseandmenthofu2ransynthase[J].Proc.Natl.Acad.Sci.USA,2001,98(15):8915-8920.[7]WangnerGJ,WangE,ShepherdRW.Newapproachesforstudyingandexploitinganoldprotuberance,theplanttrichome[J].AnnalsofBotany,2004,93:3-11.[8].[M].:,2003.[9],.cDNA[J].,2004,31(1):22-25.ConstructionofFull2lengthcDNALibraryfromTrichomesofNicotinanTabaciumCUIHong1,JIHao1,ZHANGHua1,CHENLiang23(1.NationalTobaccoCultivatePhysiologyandBiochemistryResearchCentre,HenanAgriculturalUniversity,Zhengzhou450002,China;2.SchoolofLifeSciences,XiamenUniversity,Xiamen361005,China)Abstract:Anovelcold2brushingmethodwasusedtoisolatethetrichomesfromleavesoftobaccoK326.TotalRNAwasextractedbyTrizolmethodandthefull2lengthcDNAlibrarywasconstructedusingtheSMART(switchingmechanismat5endofRNAtran2script)technology.Accordingtotheresults,therecombinationrateswas99.5%andthetiteroftheamplifiedlibrarywas1.01106pfu/mL.24positivecloneswereselectedrandomlyandsequenced,theresultsindicatedthatthesizeofinsertswasabout8001000bp.Mergingthe24sequencesand23unigeneswereobtained.Sequenceanalysisresultsindicatedthat21sequenceswerefoundthattheyhavesignificantsimilaritywithdatainGenBankand14ofthemwerethefull2lengthsequences.Theseresultsindicatedthatthelibraryisofahigh2qualitylibraryforcloningtargetgenesandforthefurtherstudyingeneexpressionprofileanalysis.Keywords:nicotinantabacium;trichome;cDNAlibrary268()2006