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ABA诱导的玉米叶片ZmCCaMK活性研究及亚细胞定位的中期报告.docx

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ABA诱导的玉米叶片ZmCCaMK活性研究及亚细胞定位的中期报告 Introduction ABA(abscisicacid)isanimportanthormonethatregulatesvariousphysiologicalprocessesinplants,includingseeddevelopment,stomatalclosure,andresponsetoenvironmentalstresses.Calcium-dependentproteinkinases(CaMKs)havebeenidentifiedaspotentialdownstreamtargetsofABAsignaling.Inmaize,ZmCCaMK(ZeamaysCalcium-calmodulin-dependentproteinkinase)isoneofthekeyCaMKsthathavebeenshowntoplayacrucialroleinABA-mediatedsignalingpathway. TheaimofthisstudyistoinvestigatetheactivityofZmCCaMKinABA-inducedmaizeleafbladesandtodetermineitssubcellularlocalizationinthemid-stage. Methods Maizeseedlingsweregrowninacontrolledenvironmentchamberwitha12-hourlightand12-hourdarkcycle.Aftertwoweeks,thesecondleavesweredetachedandtreatedwith100μMABAfordifferenttimeperiods(0,10,20,30,60,and120minutes).Thetotalproteinwasextractedfromtheleafbladesandsubjectedtoactivityassaysusingsyntheticpeptideasthesubstrate.TheactivityofZmCCaMKwasmeasuredusingaradioactivityassaywith[γ-^32P]ATP.Thephosphorylationsignalwasquantifiedbyautoradiographyandanalyzedusingaphosphorimager. TodeterminethesubcellularlocalizationofZmCCaMKinmaizeleafblades,atransientexpressionassaywasperformed.Thefull-lengthZmCCaMKcDNAwasclonedintothepCAMBIA1301-GFPvector,andthefusionproteinwasintroducedintomaizeleafbladesusingtheAgrobacterium-mediatedtransienttransformationmethod.ThelocalizationoftheGFP-taggedZmCCaMKwasvisualizedusingconfocalmicroscopy. Results TheresultsshowedthatABAtreatmentsignificantlyincreasedtheactivityofZmCCaMKinmaizeleafblades.Theactivityreacheditspeakat30minutesafterABAtreatmentandthengraduallydecreased.TheactivityofZmCCaMKinthecontrolgroupremainedalmostconstantthroughoutthetimecourse.TheseresultssuggestedthatABAeffectivelyinducedZmCCaMKactivityinmaizeleafblades. ThesubcellularlocalizationofZmCCaMKwasobservedusingconfocalmicroscopy.TheGFP-taggedZmCCaMKfusionproteinwasmainlylocatedinthecytoplasmandnucleusofthemaizecells.Noobvioussignalwasdetectedinthechloroplastormitochondria. Discussion The