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1、216PlantGenomicsinChinaⅣ水稻基因组测序及注释分析HanBingNationalCenterforGeneResearch,ChineseAcademyofSciences216PlantGenomicsinChinaⅣEstablishmentofaRiceMutantLibrarybyT-DNAInsertionChangyinWu1,WenyaYuan1,GuoxingChen1,XiangjunLi1,DongGuo1,JianZhang1,ZhihuiChen1,CaishunLi1,AndrzejKilian2,JuanLi1,Ca
2、iguoXu1,ShipingWang1andQifaZhang11NationalKeyLaboratoryofCropGeneticImprovement,NationalCenterofCropMolecularBreeding,HuazhongAgriculturalUniversity,Wuhan430070,China；2CenterfortheApplicationofMolecularBiologytoInternationalAgriculture,GPOBox3200,Canberra,ACT2601AustraliaRice(Oryzasati
3、vaL.)isanimportantcropworldwideand,withtheavailabilityofthedraftsequence,ausefulmodelforanalyzingthegenomestructureofgrasses.T-DNAtaggingisoneofthewidely-usedmethodsforgenerationofinsertionmutantsforgenefunctionalanalysisinplants.Asapartofourricefunctionalgenomicsproject,apowerfulenhan
4、certrapsystemcarryingaGAL4/VP16transactivatorandaUAS-GUSPlusreportercassettewasemployedinahighefficiencyAgrobacterium-mediatedricetransformationsystemtogeneratealibrary.Wehavecurrentlyobtainedmorethan35,000independenttransgenicplants.Thetransformantscarriedonaverage2.0copiesoftheT-DNAa
5、nd42%ofthetransformantshadsinglecopyinsertioninthismutantlibrary.GUSassayofdifferentorgansrevealedvariouspatternsofthereportergeneexpression.ContinuedscreeningforGUSactivityinleaves,rootsandflowersofT1familiesconfirmedthestabletransmissionofexpressionpatternsfromtheT0toT1generation,ind
6、icatingthatUASinricewasnotassensitivetomethylationasintobaccoandArabidopsis.ThesystemGAL4/VP16-UAS-GUSPluswillprovideforthefirsttimeapowerfultoolforthetargetedexpressionoftransgenesinanimportantmonocotyledonouscrop.TheplasmidrescueandTail-PCRstrategieswereusedtoisolatetheT-DNAflankings
7、equence.Analysisofthosesequencesfromabout1400transformantsshowedthatalmostallthesequenceshadhomologywiththesequenceinthericegenomedatabases.Wearecarryingoutphenotypicscreeningofmorethan7000T1familiestoidentifymutantsgeneratedbyT-DNAinsertionandhaveobtainedsomeconspicuousmorphological
