水稻花粉特异性基因ospsg076启动子的克隆 表达载体构建与烟草遗传转化分析-cloning and expression vector construction of rice pollen specific gene os psg 076 promoter and analysis of tobacco genetic transformation.docxVIP

AbstractBothwheatandricearethemostimportantfoodcropsintheworld.PreviousstudyshowedthatTaPSG076isapollen-specificgenethatisexpressedinthedevelopmentalprocessofpollensinwheat.However,thefunctionofTaPSGO76remainedunknown.TheresearchofPSG076genepromoterswillhelpustounderstandthegeneregulationinpollendevelopmentandthegrowthofwheat.Sincewheathashexaploidgenomeanditisverylarge,theresearchofgenesandtheirpromotersinwheatwillbetime-consumingandheavytasks,andworkefficiencyisnothigh.Bycomparison,wefoundahomologousgeneofTaPSG076inthericegenomeandthegenewasdesignatedasOsPSG076.Therefore,thepresentresearchfocusedonisolationandfunctionalanalysisofOsPSG076genepromoter,theresultsofwhichshouldbehelpfulforunderstandingtheregulatingcharacterizationofTaPSG076genepromoterinwheat.Wesuccessfullycloneda1266bpupstreamsequenceofOsPSG076fromricegenomicDNAusinginversePCR,basedonthesequenceofOsPSG076.PutativefunctionalpromoterelementswereanalyzedbythePLACEdatabase.TheresultsshowedthatthisupstreamsequencecontainedthebasicelementssuchasTATA-boxandpollenspecificcis-elementssuchasAGAAA、GTGA,indicatingthatthesemotifsmayconferpollenspecificexpressionforthepromoterdrivengene.Inordertoanalyzeexpressionspecificfunctionsofvariouscis-elementscontainedinthisOsPSG076genepromoter,sixvectorsrespectivelynamedRP13-pBI、RP10-pBI、RP7-pBI、RP4-pBI、RP3-pBI、RP2-pBI,respectively,withdifferent5-deletions:-1258bp,-986bp,678bp,-379bp,-334bpand-236bpwereinsertedintopBI121.Thesevectorswereintroducedintotobacco(NicotianatobaccumL.)plantsbyAgrobacterium-mediatedtransformationmethod.TransgenictobaccowasscreenedbyPCRusingGUSgeneanddifferentlengthsofpromoterfragmentsastargets.ResultsshowedthatonlyRP2-pBIwassuccessfullytransformedintotobacco.HistochemicalGUSstainingontransgenictobaccoplantsshowedthatthe0.2kbpromoterfragmentneitherpossessesthepollen-specificfunction,norhasthebasicpromoteractivity.Thefunctionalanalysesofotherpromoterfragmentsareunderway.Keywords:wheat,rice,OsPSG076promoter,GUSgene,tobacco,Agrobacterium-mediatedtransf