植物生物技术.docx
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植物生物技术.docx
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植物生物技术
Anauxinresponsive CLE generegulatesshootapicalmeristemdevelopmentin Arabidopsis
Planthormoneauxinregulatesmost,ifnotallaspectsofplantgrowthanddevelopment,includinglateralrootformation,organpattering,apicaldominance,andtropisms.Peptidehormonesarepeptideswithhormoneactivities.Someofthefunctionsofpeptidehormonesinregulatingplantgrowthanddevelopmentaresimilartothatofauxin,however,therelationshipbetweenauxinandpeptidehormonesremainslargelyunknown.Herewereporttheidentificationof OsCLE48,arice(Oryzasativa) CLE (CLAVATA3/ENDOSPERMSURROUNDINGREGION)gene,asanauxinresponsegene,andthefunctionalcharacterizationof OsCLE48 in Arabidopsis andrice. OsCLE48encodesaCLEpeptidehormonethatissimilarto Arabidopsis CLEs.RT-PCRanalysisshowedthatOsCLE48 wasinducedbyexogenouslyapplicationofIAA(indole-3-aceticacid),anaturallyoccurredauxin.Expressionofintegrated OsCLE48p:
GUS reportergeneintransgenic Arabidopsis plantswasalsoinducedbyexogenouslyIAAtreatment.Theseresultsindicatethat OsCLE48 isanauxinresponsivegene.Histochemicalstainingshowedthat GUS activitywasdetectedinallthetissueandorgansofthe OsCLE48p:
GUS transgenic Arabidopsis plants.Expressionof OsCLE48 underthecontrolofthe 35S promoterin Arabidopsis inhibitedshootapicalmeristemdevelopment.Expressionof OsCLE48 underthecontrolofthe CLV3 nativeregulatoryelementsalmostcompletelycomplemented clv3-2 mutantphenotypes,suggestingthatOsCLE48isfunctionallysimilartoCLV3.Ontheotherhand,expressionof OsCLE48 underthecontrolofthe 35S promoterin Arabidopsis haslittle,ifanyeffectsonrootapicalmeristemdevelopment,andtransgenicriceplantsoverexpressingOsCLE48 aremorphologicallyindistinguishablefromwildtypeplants,suggestingthatthefunctionsofsomeCLEpeptidesmaynotbefullyconservedin Arabidopsis andrice.Takentogether,ourresultsshowedthat OsCLE48 isanauxinresponsivepeptidehormonegene,anditregulatesshootapicalmeristemdevelopmentwhenexpressedin Arabidopsis.
Introduction
Auxinregulatesmanyaspectsofplantgrowthanddevelopment,includingapicaldominance,organformation,lateralrootformation,rootandstemelongation,andvasculardevelopment(Davies,1995).Itisverylikelythatauxinregulatedplantgrowthanddevelopmentisinitiatedbytheactivationofauxinresponsegenesbylocallyincreasedauxinconcentration(ChapmanandEstelle,2009).
Activationofauxinresponsegenesiscontrolledbytwodifferentgroupsoftranscriptionfactors,auxinresponsefactors(ARFs)andAux/IAAproteins.Whenthecellularauxinlevelislow,Aux/IAAproteinsaredimmerizedwithARFs,thusinhibittheexpressionofauxinresponsegenes.Whentheauxinleveliselevated,auxinbindstoTIR1auxinreceptor,leadingtotheactivationofTIR1,andeventuallythedegradationofAux/IAAproteinsby26Sproteasome,resultintheactivationofauxinresponsegenesbyARFs(Dharmasirietal.,2005; KepinskiandLeyser,2005; GuilfoyleandHagen,2007; Tanetal.,2007; Hayashi,2012).
Severaldifferentgenefamiliesincluding Aux/IAAs, GH3s,and SAURs havebeenidentifiedasauxinresponsegenes(HagenandGuilfoyle,2002).Expressionofsomeothergenessuchas ASL/LBD(LATERALORGANBOUNDARIESDOMAIN/ASYMMETRICLEAVES2-LIKE)isalsoinducedbyauxin(Leeetal.,2009; Coudertetal.,2013).However,consideringthatauxinregulatesalmosteveryaspectofplantgrowthanddevelopment,itislikelythatlargenumbersofauxinresponsegenesremainunidentified(Kiefferetal.,2010).
Peptidehormonesarepeptideswithhormoneactivities.Peptidehormonesregulatemanycellularprocessesinanimals,bacteriaandyeast(EdlundandJessell,1999).Ithasbeenbelievedthatplantsdonotproducepeptidehormones,untiltheidentificationofSystemin,a18aminoacidspeptide,asasignalmoleculeinwoundingresponseintomato(Pearceetal.,1991).Sofarnearly20plantpeptidehormoneshavebeenidentified(Germainetal.,2006; Hashimotoetal.,2008; Katsiretal.,2011;LeasureandHe,2012).
Plantpeptidehormonesareencodedbygeneswithsmallopenreadingframes(ORFs),andcanbeclassifiedintotwodifferentgroups:
secretedandnon-secretedpeptidehormones(Hashimotoetal.,2008; Katsiretal.,2011; Matsubayashi,2011).PrecursorsofsecretedpeptidehormonescontainanN-terminalsignalpeptide,whichfacilitiesproteintransportandwillberemovedduringpost-transcriptionalmodifications.Ontheotherhand,non-secretedpeptidehormonesdonotcontainsignalpeptides(Hashimotoetal.,2008; Katsiretal.,2011; Matsubayashi,2011).
Non-secretedplantpeptidehormonesincludeSystemin,Enod40(EarlyNodulin40),POLARIS(PLS),ROTFOURLIKE(RTFL),amdBRICK1(BRK1; Charonetal.,1997; Germainetal.,2006;Hashimotoetal.,2008).Non-secretedpeptidehormonesareinvolvedintheregulationofplantgrowthanddevelopment,andplantresponsetoenviromentalstresses.Forexample,PLSregulatesrootgrowthandvasculardevelopment(Cassonetal.,2002),RTFLpeptidesDEVIL(DVL1)andROTUNDIFOLIA4(ROT4)regulateleafandfruitdevelopment(Naritaetal.,2004; Wenetal.,2004),IDLpeptidesregulatefloralorganabscission(Butenkoetal.,2003; Choetal.,2008; Stenviketal.,2008),andSystemininvlovesintheregulationofplantstressresponse(Pearceetal.,1991,2001; Constabeletal.,1998).
Morethan10differentsecretedpeptidehormoneshavebeenidentifiedinplants,includingCLAVATA3/ENDOSPERMSURROUNDINGREGION(CLE),EPIDERMALPATTERNINGFACTOR(EPF),ROOTMERISTEMGROWTHFACTOR(RGF)/CLELIKE(CLEL)/GOLVEN(GLV),INFLORESCENCEDEFICIENTINABSCISSIONLIKE(IDL),andRAPIDALKALINISATIONFACTOR(RALF).Secretedpeptidehormonesaremainlyinvolvedintheregulationofplantgrowthanddevelopment.Forexample,CLEpeptidesareinvolvedintheregulationofshootandrootapicalmeristem(SAMandRAM)mataining(Kinoshitaetal.,2007; Junetal.,2010; Katsiretal.,2011),RGF/CLEL/GLVpeptidesregulaterootgrowthandlateralrootformation(Matsuzakietal.,2010; Mengetal.,2012a; Fernandezetal.,2013),andEPFpeptidesregulatestomatadevelopment(Haraetal.,2007; HuntandGray,2009; Suganoetal.,2010).Mostofthesecretedpeptidehormonesareencodedbygenefamilies.In Arabidopsis,forexample,thereare32genesencodingCLEpeptides,11encodingRGF/CLEL/GLVpeptides,andsixencodingIDLpeptides(Sawaetal.,2008; Stenviketal.,2008; Matsuzakietal.,2010; Mengetal.,2012a).
Verylimitedexperimentalevidencehasshownthatthereiscross-talkbetweenauxinandsomeofthepeptidehormones.Forexample,PLSandRGF/CLEL/GLVpeptideshavebeenshowntoregulateauxintransport,andtheexpressionof PLS and RGF/CLEL/GLV genesareregulatedbyauxin(Cassonetal.,2002; Chilleyetal.,2006; Mengetal.,2012b; Whitfordetal.,2012).Ontheotherhand,auxinhasbeenshowntoinvolveinCLE-inducedvascularproliferation(Whitfordetal.,2008).Inthisstudy,wereporttheidentificationof OsCLE48,arice CLE geneasanauxinresponsegene,andthefunctionalcharacterizationof OsCLE48 in Arabidopsis andrice.
MaterialsandMethods
PlantMaterialsandGrowthConditions
The Arabidopsisthaliana (Arabidopsis)ecotypeColumbia(Col-0)andJaponicarice(Oryzasativa)variety Nipponbare wereusedforplanttransformation. Arabidopsis mutant clv3-2 isintheecotypeLandsbergerecta (Ler)background(Clarketal.,1995).
Riceseedsweregerminatedandgrownonwaterfor10days.Seedlingswerethentransferredintosoilpotsandkeptinagrowthroom. Arabidopsis seedsweresterilizedandgrownonplatescontaining½MS(MurashigeandSkoog)mediumwithvitamins(PlantMedia)and1%(w/v)sucrose,solidifiedwith0.6%(w/v)phytoagar(PlantMedia).Seedlingsweretransferredintosoilpotsandkeptinagrowthroom.Forplanttransformationandphenotypicanalysisofadultplants, Arabidopsis seedsweresowndirectlyintosoilandgrowninagrowthroom. Arabidopsis plantsweregrownat20°C,andriceplantsat28°C,witha16hlight/8hdarknessphotoperiod.
RNAIsolationandRT-PCR
TotalRNAfromricewasisolatedfollowingtheproceduredescribedpreviouslyforRNAisolationfrompoplar(Geraldesetal.,2011; Wangetal.,2014).TotalRNAfrom Arabidopsis wasisolatedusingtheEazyPurePlantRNAKit(TransGenBiotech)followingthemanufacturer’sprocedure.cDNAwassynthesizedusingtheEazyScriptFirst-StrandDNASynthesisSuperMix(TransGenBiotech)accordingtothemanufacturer’sinstructions.RT-PCRorquantitativeRT-PCRwasusedtoexaminetheexpressionofcorrespondinggenes.QuantitativeRT-PCRwasperformedonaStepOneReal-TimePCRsystem(AppliedBiosystems)withStepOneSoftwarev2.1.Allreactionswereperformedinthreereplications.Expressionof Arabidopsis gene ACTIN2 (ACT2)orricegeneOsACT2 wasusedascontrolforRT-PCR.Ricegene UBQ5 (Jainetal.,2006)wasusedascontrolreferencegeneforquantitativeRT-PCR.
AllprimersusedinthisstudyincludingprimersforgenecloningandprimersforgeneexpressionanalysiswerelistedinTable 1.
TABLE1
TABLE1.Listofprimersusedinthisstudy.
Constructs
TogenerateHAtagged OsCLE48 constructforplanttransformation,the252bpfull-lengthORFofOsCLE48 wasamplifiedbyRT-PCRusingRNAisolatedfrom10-days-oldriceseedlings,andclonedinframewithanN-terminalHAtagintothe pUC19 vectorunderthecontrolofthedouble 35Senhancerpromoterof CaMV.The pUC19OsCLE48 constructgeneratedwasthendigestedwithproperenzymes,andsubclonedintovector pPZP211 and pCAMBIA1301 togeneratebinaryvectorpPZP211OsCLE48 and pCAMBIA1301OsCLE48 for Arabidopsis andricetransformation,respectively.
Togeneratethe
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