烟草幼苗两种钙调素结合蛋白激酶的活性调节及基因表达

305,JournalofPlantPhysiologyandMolecularBiology2005,31(3):3053102004-07-082005-03-17(No.30070078)*E-mail:[e-mail];Tel:[phone]*222(14300622430072)(NtCBK1NtCBK2)pH7.58Mg2+Na+NorthernNtCBK1NtCBK2;NtCBK1NtCBK1NorthernQ945/(HeplerWayne1985;Sanders2002)CDPK(calcium-dependentproteinkinase)CDPKCa2+(Harmon2000,2001;Harper2004)CDPKCDPKCaMK(calcium/calmodulin-dependentproteinkinase)Ca2+/CaMCDPKCaMKII(Schulman1993;Koyama1999)CaMKCaMKIICDPKCaMK(ZhangLu2003)Watillon(1993)CaMKIIcDNACaMKIICaMMCK1(Lu1996;Wang2001)MCK1OsCBKNtCBK1/2CaMCaMOsCBKCaMNtCBK1Ca2+CaMCaMNtCBK1NtCBK2Ca2+CaMCaM(Zhang2002;Hua2003,2004a)(ZhangLu2003)Ca2+/CaM/CBK30631(Ali2003)(SneddenFromm1998;YangPoovaiah2003)NtCBK1NtCBK1NtCBK1NtCBK2CBKpHNtCBK1NtCBK2NtCBK1NtCBK2(Hua2004a)NAAABAGANtCBK2NtCBK111.1(NicotianatabacumL.W38)30d28~3016h42410%PEG-60000.1mmol/LGANAAABA300mmol/LNaCl(Hua2004b)100mgRNA1.2()(Hua2003,2004a)10000×g10minNi-NTA4~10Buffer(20mmol/LTris-HClpH8.5;100mmol/LKCl;100mmol/L;10mmol/Lβ-;10%)1.33025mmol/LTris-HClpH7.50.5mmol/L(DTT)10mmol/L100µmol/LATP3.7×105Bq[γ-32P]ATP1µmol/LCaM/1mmol/LCaCl2()NtCBKs30min1/55×SDS-PAGE10%SDS-PAGE0.1%30min100%(Zhang2002)1.4RNANorthern1mLTRizol5min0.2mL15s2~3min12000×g415min0.5mLRNA10min12000×g410min70DEPC-H2ORNARNA653'(Promega)(Hua2004b)22.1pHMg2+Na+NtCBK1pHNtCBK1pH7.5pHpH6.0NtCBK140%pH10.0,NtCBK145%(1A)Mg2+NtCBK1Mg2+5~10mmol/L(1B)Na+Na+(1C)307:32.2pHMg2+Na+NtCBK2pHMg2+Na+NtCBK2NtCBK2pHNtCBK2pH8.0pH(2A)Mg2+Mg2+Mg2+Mg2+10mmol/LMg2+NtCBK2(2B)Na+NtCBK2(2C)2.3NtCBK1/2NtCBK13'NtCBK1NorthernNtCBK1(3A)NtCBK2(3B)2.4NtCBK1NtCBK1442300mmol/LNaCL10%PEG-600030d0.1mmol/LABANAAGA30d2pH(A)Mg2+(B)Na+(C)NtCBK2Fig.2AutophosphorylationofNtCBK2indifferentpH(A),Mg2+concentrations(B)orNa+concentrations(C)1pH(A)Mg2+(B)Na+(C)NtCBK1Fig.1AutophosphorylationofNtCBK1atdifferentpH(A),Mg2+concentrations(B)orNa+concentrations(C)3NtCBK1(A)NtCBK2(B)NorthernblotFig.3NorthernblotanalysisoftheexpressionofNtCBK1(A)orNtCBK2(B)intobaccoorgans1:Root;2:Stem;3:Leaf;4:Flower;5:Seed.308310~240minRNA20µgNorthernNtCBK1(4A);ABANAAGANtCBK1(4BC);300mmol/LNaCl10minNtCBK130minNtCBK15(4C)3Ca2+/CaM/CBKCaMCaM(Takezawa1995;Yamakawa2001)CaM(Liao1996;Lee2000;Takezawa1996)CaMCa2+(NtCaM1/3/13)NtCBK2NtCBK13NtCBK1(Hua2003,2004a)CaMCaMCa2+pHNtCBK2pHNtCBK2pH8.0pH(2A);NtCBK1pH7.5pH(1A)CDPKCDPK(Lee1998;Harmon1987)CDPKpHpH(6.0~9.0)pHpH(9.0~13.0)pHpHMg2+Na+Mg2+(1B2B)Mg2+ATPMg2+ATPATP(Hers1952)Na+(1C2C)NtCBK1/24NtCBK1Fig.4TheexpressionofNtCBK1undertemperature,droughtandhormoneA:4and42;B:0.1mmol/LNAAandGA;C:300mmol/LNaCl,10%PEG-6000and0.1mmol/LABA.309:3NtCBK1/2(3);NAAABA(4)NtCBK1NtCBK2NtCBK2NtCBK1NtCBK2GANtCBK2NtCBK1(Hua2004a)NtCBK1NtCBK2NtCBK2GANtCBK1NtCBK2CDPKCDPK(Urao1994;Saijo2000)NtCBK1NtCBK1NtCBK1NtCBKsNtCBKs(CDPKs/CBK/CCaMK)AliGS,ReddyVS,LindgrenPB,JakobekJL,ReddyASN(2003).Differentialexpressionofgenesencodingcalmodulin-bindingproteinsinresponsetobacterialpathogensandinducersofdefenseresponses.PlantMolBiol,51:803815HarmonAC,GribskovM,HarperJF(2000).CDPKakinaseforeveryCa2+signal?TrendsPlantSci,5:154159HarmonAC,GribskovM,GubriumE,HarperJF(2001).TheCDPKsuperfamilyofproteinkinases.NewPhytol,151:175183HarmonAC,Putmen-EvensC,CormierMJ(1987).Acalcium-dependentbutcalmodulin-independentproteinkinasefromsoybean.JPlantPhysiol,83:830837HarperJF,BratonG,HarmonA(2004).DecodingCa2+signalthroughplantproteinkinases.AnnuRevPlantBiol,55:263288HeplerPK,WayneRO(1985).Calciumandplantdevelopment.AnnuRevPlantPhysiolPlantMolBiol,36:397439HersHG(1952).Roleofmagnesiumandpotassiuminthereactionfructokinasique.BiochimBiophysActa,8:424430HuaW,LiRJ,WangL,LuYT(2004a).Atobaccocalmodulin-bindingproteinkinase(NtCBK2)inducedbyhigh-salt/GAtreatmentanditsexpressionduringfloraldevelopmentandembryogenesis.PlantSci,166:12531259HuaW,LiangSP,LuYT(2003).Atobaccocalmodulin-bindingproteinkinase,NtCBK2,isregulateddifferentiallybycalmodulinisoforms.BiochemJ,376:291302HuaW,ZhangL,LiangSP,JonesRL,LuYT(2004b).Atobaccocalmodulin-bindingproteinkinasefunctionsasanegativeregulatorofflowering.JBiolChem,279:3148331494KoyamaM,SpicerSS,SchulteBA(1999).ImmunohistochemicallocalizationofCa2+/calmodulin-dependentproteinkinaseinouterhaircells.JHisCyt,47:712LeeJY,YooBC,HarmonAC(1998).Kineticandcalcium-bindingpropertiesofthreecalcium-dependentproteinkinaseisoenzymesfromsoybean.Biochemistry,37:68016809LeeSH,JohnsonJD,WalshMP,vanLieropJE,SutherlandC,XuA,SneddenWA,Kosk-KosickaD,FrommH,NarayananN,ChoMJ(2000).DifferentialregulationofCa2+/calmodulin-dependentenzymesbyplantcalmodulinisoformsandfreeCa2+concentration.BiochemJ,350:299306LiaoB,GawienowskiMC,ZielinskiRE(1996).DifferentialstimulationofNADkinaseandbindingofpeptidesubstratesbywild-typeandmutantplantcalmodulinisoforms.ArchBiochemBiophys,327:5360LuYT,HidakaH,FeldmanLJ(1996).Characterizationofacalcium/calmodulin-dependentproteinkinasehomologfrommaizerootsshowinglight-regulatedgravitropism.Planta,199:1824SaijoY,HataS,KyozukaJ,ShimamotoK,IzuiK(2000).Over-expressionofasingeCa2+-dependentproteinkinaseconfersbothcoldandsalt/droughttoleranceonriceplants.PlantJ,23:319327SandersD,PellouxJ,