AcDs(GUS)结构介导的水稻启动子捕获系统的建立

575,JournalofPlantPhysiologyandMolecularBiology2005,31(6):5755802005-03-152005-09-02863(No.2002AA2Z1003)*(E-mail:[e-mail];Tel:[phone])Ac/Ds(GUS)1,21334411*(1200032222111632011064200237)Activator/Dissociation(β-glucuronidase)[Ac/Ds(GUS)]p13B1118T2141PCR37DsDs5GUSAc/DsGUSBarQ74(430Mbp)(Harushima1998RGP:)(Yu2005;Goff2002;IRGP2005;Sasaki2002;Feng2002;Thericechromosome10sequenceingconsortium2003)28000cDNA(Kikuchi2003)T-DNAAc/Ds(Jeong2002Kolesnik2004)(genetrap)(DunnHandelsman1999;Chelstowska1999;LukacsovichYamamoto2001;Lindsey1993)GUSDsDs(2003)BarDsBar11.111(OryzasativaL.subsp.japonicacv.Zhonghua11)1.2EHA105pCAMBIA1300Jefferson()UbipHM2Ac(Ts)pUbiTsDspDsBar13001.3(1998)12~15d2,4-DN64dp13B(EHA105/p13B)N65763121.4DNA2cm200µLTPS(Tris-HCl100mmol/L,EDTA10mmol/L,KCl1mmol/L,pH8.0)7520min14000×g5min120µL120µL14000×g10min300µL70DNA30µLTEDNA1.5PCR1P1(5'-ctagccttttcatgggagtg-3'Bar5')P2(5'-cagctgccagaaacccacgtc-3'Bar3')P3(5'-ttagccctgccttcatacg-3'Ubi)DsDs6~7kbDs340bp(P1P3)1100bp(P2P3)DNAP3P4(5'-cgtcatctgaggaatggagtc-3'Ac)DsDs420bpDsDs9445s5840s721min307210min10µL1.6Basta2Datta(1992)Basta3000.1%SDS2~32d32(2000)1.7GUSJefferson(1987)22.1p13Bp13BpCAMBIA1300T-DNADsGUSAc(Actransposase)DsGUSDsAcDsDsUbi(ubiquitons)DsBar()UbiAcBarGUS(1)()2.2T2Basta(1998)p13B1118200318T2Ds18T2(1)2~3Basta3(459)9010(1~3610~15)502(816)0.1%3(71718)(1)1020173Basta141Basta81.52A8T21p13BFig.1Schematicdiagramofplasmidp13BBar:CodingregionofBargene;Ds:TheleftorrightborderofDselement;GUS:CodingregionofGUSreportergene;N:Nosterminator;AcTSase:Codingregionoftransposase;UbiPro:Promoterofubiquitons.ArrowsindicatethepositionsofPCRprimers.577:Ac/Ds(GUS)6Basta2B2ABasta2.3BastaDsBastaDsBastaDNAP1P3PCR141126340bp(3A1)DsDs(2003)126DsDsP3P4PCR1T2DsTable1Summaryofherbicidesreening,DstranspositionandhistochemicalstainingofT2generationoftransgenicplantsCodeofNumber*Percentageofherbicide-NumberofNumberofherbicide-NumberofNumbertransgenicofseedsresistantplantsobtainedfurtherscreenedresistantplantsobtainedplantswithDsofGUS+linestested(g)byspraying(%)plantsbyfurtherscreeningtranspositionplants18550201500280508600361508611480901010305809010900661.5501030076100000848.6Approx.0.1654398090101020106650104001185502020001280502018140136550161660144350107611580501110101660Approx.0.142001780000001863.400000Total173141375*Theweightof200grainsofriceisapproximately4.3gram.28T2BastaFig.2ScreeningofT2generationofNo.8transgeniclinewithBastaA:TransgenicplantsonwhichBastahadbeensprayed;B:LeavesoftransgenicplantsonwhichBastahadbeensprayed.5783131420bp(3B)31Ds420bpDsT-DNADs9P1P3P3P4DsBar3'P2(Ds1050bp)P3PCR61100bp(3C)DNADsDNA37Ds6T32~4BastaPCRT2()37Ds(1)2.4DsT2GUST2Ds375GUS(1)4GUSDsGUS3TAILPCRInversePCR(Wei1997;Casson2002;Lee2004)4DsGUSFig.4HistochemicalstainingofdifferentorgansortissuesoftransgenicplantsinwhichDshavebeentransposedA:Stamen,pistilandovary;B:Hull;C:Ovary;D:Embryoandendosperm;E:Endosperm.3PCRFig.3ElectrophoresisofPCRproductsofBasta-resistanttransgenicplants(A)ThePCRproductsobtainedbyusingprimersP1andP3.M:100bpladdermarker;Lane1:plasmidp13B;Lane2:Zhonghua11;Lanes312:Basta-resistanttransgenicplants.(B)ThePCRproductsobtainedbyusingprimersP3andP4.M:100bpladdermarker;Lane1:plasmidp13B;Lane2:Zhonghua11;Lanes312:Basta-resistanttransgenicplantssameasthosein(A).(C)ThePCRproductsobtainedbyusingprimersP2andP3.M:100bpladdermarker;Lane1:plasmidp13B;Lane2:Zhonghua11;Lanes39:PCRproductsofBasta-resistanttransgenicplantswhicharepresent(lane3)orabsent(lanes49)in(B).579:Ac/Ds(GUS)6Ac/DsAc/Ds(2001)Ac/DsAcDsAcDsDsDsDsAcDsAcT-DNAUbiDsAcDsDsDs(2003)DsDsBarDsUbiBarBarDsAc/DsAc/Dsp13Bp13B()Ac/Ds(Ito2002)T-DNA()T-DNADsTrapDsDsGUST3CassonSA,ChilleyPM,ToppingJF,EvansIM,SouterMA,LindseyK(2002).ThePOLARISgeneofArabidopsisencodesapredictedpeptiderequiredforcorrectrootgrowthandleafvascularpatterning.PlantCell,14(8):1705–1721ChelstowskaA,LiuZ,JiaY,AmbergD,ButowRA(1999).SignallingbetweenmitochondriaandthenucleusregulatestheexpressionofanewD-lactatedehydrogenaseactivityinyeast.Yeast,15(13):1377–1391ChenY(),ChengSJ(),WangJ(),ShenGZ(),AnLS(),ZhangJL()(2000).ArapidandsimplemethodforassayingPPTresistanceintransgenicriceplants.PlantPhysiolCommun(),36(1):50–52(inChinese)DattaSK,DattaK,SoltanifarN,DonnG,PotrykusI(1992).Herbicide-resistantindicariceplantsfromIRRIbreedinglineIR72afterPEG-mediatedtransformationofprotoplasts.PlantMolBiol,20:619–629DunnAK,HandelsmanJ(1999).AvectorforpromotertrappinginBacilluscereus.Gene,226:297–305FengQ,ZhangY,HaoP,WangS,FuG,HuangY,LiY,ZhuJ,LiuY,HuXetal.(2002).Sequenceandanalysisofricechromosome4.Nature,420:316–320GoffSA,RickeD,LanTH,PrestingG,WangR,DunnM,GlazebrookJ,SessionsA,OellerP,VarmaHetal.(2002).Adraftsequenceofthericegenome(Oryzasativassp.japonica).Science,296(5565):92–100HarushimaY,YanoM,ShomuraA,SatoM,ShimanoT,KubokiY,YamamotoT,LinSY,AntonioBA,ParcoAetal.(1998).Ahigh-densityricegeneticlinkagemapwith2275markersusingasingleF2population.Genetics,148:479–494InternationalRiceGenomeSequencingProject(2005).Themap-basedsequenceof