水稻基因OsASIE1 抗逆功能分析

作物学报ACTAAGRONOMICASINICA2011,37(10):1771−1778:[e-mail](2008ZX001-003,2009ZX08009-007B)*(Correspondingauthor):,E-mail:[e-mail],Tel:[phone]:E-mail:[e-mail],Tel:[phone]Received():2011-03-14;Accepted():2011-06-25;Publishedonline():2011-07-28.URL::10.3724/SP.J.1006.2011.01771水稻基因OsASIE1抗逆功能分析吴慧敏黄立钰潘雅娇靳鹏傅彬英*/,100081:,AP2/EREBPAP2/EREBPEREBPOsASIE1(abioticstressinducedEREBPgene),OsASIE1(electrophoresismobilityshiftassay,EMSA),AP2DRE(dehydration-responsiveelement)GCCbox(ethyleneresponseele-ment),OsASIE1DREGCCbox,:;AP2/EREBP;EMSA;;FunctionAnalysisofGeneOsASIE1RespondingtoAbioticStressesinRiceWUHui-Min,HUANGLi-Yu,PANYa-Jiao,JINPeng,andFUBin-Ying*InstituteofCropSciences/NationalKeyFacilityforCropGeneResourcesandGeneticImprovement,ChineseAcademyofAgriculturalSciences,Beijing100081,ChinaAbstract:AP2/EREBPtranscriptionfactorsplayanimportantroleinplantdevelopment,hormoneresponse,bioticandabioticstressresponses.WeidentifiedthatOsASIE1,amemberofEREBPsubfamilyofAP2/EREBPtranscriptionfactorfamilyinrice,wasinvolvedinabioticstressresponse.TheexpressionofOsASIE1wasinducedbydroughtandsaltstresses,andover-expressionofOsASIE1inthetransgenicriceplantcouldimproveitstolerancetosaltstress.Furtherelectrophoresismobilityshiftassay(EMSA)revealedthattheAP2domainofOsASIE1proteincouldbindbothDRE(dehydration-responsiveelement)andGCCbox(ethyleneresponseelement,ERE)invitro.AlltheseresultsimplicatedthatOsASIE1mightbeinvolvedinabioticstressresponsebyregulatingtheexpressionofdownstreamgeneswithDREandGCCboxbinding.Keywords:Rice;AP2/EREBPtranscriptionfactor;EMSA;Salttolerance;Over-expression,,,[1]AP2/EREBPbZIPNACMYBWRKY[2]AP2/EREBP60AP2,DNAAP2,AP2/EREBP:AP2EREBPRAV4AP22AP2,RAVAP2B3;EREBPAP2,ERFDREB/CBF[3-4]2,EREBPs177237ERFs,[1,5]ERF4(pathe-genesis-related,PR)EREBP1-4,GCCboxGCCboxPR5′UTR,AGCCGCC[6]1997,Zhou3ERF(Pti-4Pti-5Pti-6)Pto[7-8],ERFsERFs,ERF1Pti4ATERF1[5]ERF[9][10-11][12]DREBs,ABADREBsDRE/CRTDRE(dehydration-responsiveelement)rd29A,A/GCCGAC[13]DRECRT(C-repeat)cor15a,TGGCCGA[14-15]DRE/CRT[16]DREB[13,17-18],DREBs[19]DREBOsDREB1A-GOsDREB2AOsDREB2B,OsDREB1AOsDREB1EOsDREB1GOsDREB2AOsDREB2BDRE[17-18]OsDREB1A[17];OsDRE-B1GOsDREB2B,OsDREB1E[18]ERFsDREBsEREBPsDRE,GCCbox,TINY[20]TINY2[21]BnDREBIII[22]AtERF1AtERF4AtEBPCBF1[23];DRE,CBF2/DREB1CCBF3/DREB1AERFsGCCbox,DREBsDRE,Sakuma[3]DREBsAP21520(Val,V)(Glu,E),ERFsAP21520(Ala,A)(Asp,D),,Val-15Glu-20Val-15DRE,GCCbox,ERFsDREBsGCCbox,[20]2DRE2AP216,Ser-16DREBTINYGCCboxTINY,,DREGCCboxAP2/EREBP[24],DREBsERFs,EREBPs,EREBPsEREBPs,EREBPsOsASIE1(Os08g0408500),GCCboxDRE,,1材料与方法1.1(Nipponbare,Oryzasativassp.japonica)IRAT109FL478(LTH)1.21.2.1水稻品种日本晴的低温、高盐和干旱胁迫,373d,10:OsASIE11773,2,,Yoshida(4)(150mmolL−1NaCl)(20%PEG),0h0.5h3h6h12h24h,,−701.2.2抗逆水稻品种的低温、高盐和干旱胁迫,(IRAT109),(LTH)(FL478)()(150mmolL−1NaCl,48h)(4,48h),,,−701.3PCRTRIZOLRNA[24]PCR,OsASIE1PCRF:5′-TGGTCTGATTTGGTAGCC-3′;R:5′-TCCAAGAACTGGCAGACGA-3′;Actin1F:5′-GACTCTGGTGATGGTGTCAGC-3′;R:5′-GGCTGGAAGAGGACCTCAGG-3′1.4(EMSA)1.4.1原核表达蛋白的制备PCROsASIE1AP2(67~193aa)pET-32a(Novagen,USA,69015-3),BL21(DE3)pLysS(TransGenBiotech,China,Cat.No:DR101),His-trap(GE),Miliproe,Bradford[31]1.4.2目的DNA序列及突变序列的制备GCCbox(CATAAGAGCCGCCACT)(CATAAGATCCTCCACT),DRE(ATACTACCGACATGAG)(ATACTACTGATATGAG)3,(100nmolL−1),9510min,1.4.3EMSA实验InvitrogenEMSA1.5BioEdit7.0OsASIE1EREBPMEGA4OsASIE1EREBP,(Neighbor-JoiningMethod)[25]CompleteDeletion,Bootstrap1.6OsASIE11.6.1载体构建和遗传转化,IRAT109OsASIE1cDNA,5′3′PstIBamHI,F:5′-GCACTGCAGATGGCAGCTGCTATAGAAGG-3′;R:5′-TAAGGATCCTTATTGTTGTTGAGCAGC-3′,pCUbi1390(UbIquitin1)[26],(EHA105),,1.6.2转基因植株表型鉴定T1(100mgL−1)1/2MS,96PCR,Yoshida,4150mmolL−1NaCl3,2结果与分析2.1OsASIE12.1.1日本晴中OsASIE1在低温、高盐和PEG胁迫下的表达量变化谱OsASIE1,PCR3OsASIE1,(1)PEG,OsASIE1,3h,PEGOsASIE1,OsASIE1,,20OsASIE1,,,,,OsASIE1,[12],2.1.2OsASIE1在典型抗逆水稻品种中胁迫前后的表达差异OsASIE1,OsASIE1,OsASIE1177437,(2),OsASIE1,,OsASIE1;,OsASIE12.2OsASIE1AP2GCCboxDREEREBPsGCCboxDRE,,[6,13](EMSA)OsASIE1AP2GCCboxDRE,OsASIE1AP2GCCboxDRE,(3)OsASIE1GCCboxDRE,2.3OsASIE1EREBPOsASIE1AP2GCCboxDRE,EREBPOsASIE1(4),OsASIE1DREBsOsASIE115(Val),DREB,20(Glu)(Leu),Os-DREB1AOsDREB1B20(Glu)(Val),OsASIE1GCCboxDREAtERF1AtERF4AtEBP16(Ser,S),AP2/EREBP2.4OsASIE1OsASIE1图1以实时定量PCR分析OsASIE1在非生物胁迫下的表达量差异Fig.1Real-timePCRanalysisofrelativeexpressionofOsASIE1underabioticstresses:4;:150mmolL−1NaCl;:20%PEG00.51361224hPCRColdstressistreatedat4,saltstressistreatedunder150mmolL−1NaCl,osmoticstressistreatedin20%PEG.Leafsampleswerecol-lectedat0,0.5,1,3,6,12,and24hoftreatmentforRT-PCRanalysis,respectively.图2以实时定量PCR分析OsASIE1在典型抗逆水稻品种中胁迫前后的表达量差异Fig.2Real-timePCRanalysisoftheexpressionofOsASIE1understressinricevarietieswithdifferentabioticstresstolerancesA:OsASIE1;B:OsASIE1FL478;C:OsASIE1IRAT109N:;L:;F:FL478;I:IRAT109A:ExpressionchangesofOsASIE1undercoldstressinLTHandNipponbare;B:ExpressionchangesofOsASIE1undersaltstressinFL478andNipponbare;C:ExpressionchangesofOsASIE1underdroughtstressinIRAT109andNipponbare.N,L,I,andFi