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1、作物學(xué)報ACTAAGRONOMICASINICA2016,42(7):1094?1099http://zwxb.chinacrops.org/ISSN0496-3490;CODENTSHPA9E-mail:xbzw@chinajournal.net.cnDOI:10.3724/SP.J.1006.2016.01094花生AhDGAT2a基因啟動子的克隆和功能驗(yàn)證1,21,31,21,211鄭玲史靈敏田?，搯卫走呾彻?1,2,*1,2,3,*宣寧萬書波彭振英12山東省農(nóng)業(yè)科學(xué)院生物技術(shù)研究中心/山東省作物遺
2、傳改良與生態(tài)生理重點(diǎn)實(shí)驗(yàn)室,山東濟(jì)南250100;山東大學(xué)生命科學(xué)學(xué)院,3山東濟(jì)南250100;新疆農(nóng)業(yè)大學(xué)農(nóng)學(xué)院,新疆烏魯木齊830000摘要:二酰甘油?；D(zhuǎn)移酶(DGAT)是三酰甘油(TAG)合成途徑的限速酶,對脂肪酸合成的調(diào)節(jié)具有關(guān)鍵作用。為了研究AhDGAT2a的表達(dá)調(diào)控,利用GenomeWalking方法從魯花14基因組中克隆了AhDGAT2a上游5?側(cè)翼調(diào)控區(qū)1200bp序列,即AhDGAT2a啟動子(pAhDGAT2a)序列,并利用生物信息學(xué)軟件分析其包含的調(diào)控元件,發(fā)現(xiàn)其含有多個TATA-b
3、ox和CAAT-box、光調(diào)控元件、脅迫防御相關(guān)元件和激素響應(yīng)元件。用pAhDGAT2a構(gòu)建pAhDGAT2a:GUS植物表達(dá)載體并轉(zhuǎn)化煙草品種SR1。利用組織染色法鑒定轉(zhuǎn)基因煙草的GUS表達(dá)模式,發(fā)現(xiàn)在轉(zhuǎn)基因煙草的各個器官均有GUS酶活,在柱頭、花藥和幼嫩種子中表達(dá)量較高,說明pAhDGAT2a具有一定的組成型啟動子活性。關(guān)鍵詞:花生;AhDGAT2a基因;啟動子;功能驗(yàn)證CloningandFunctionalAnalysisofPeanutAhDGAT2aPromoter1,21,31,21,2111
4、ZHENGLing,SHILing-Min,TIANHai-Ying,SHANLei,BIANFei,GUOFeng,XUANNing,1,2,*1,2,3,*WANShuo-Bo,andPENGZhen-Ying1BiotechnologyResearchCenter,ShandongAcademyofAgriculturalSciences/ShandongProvincialKeyLaboratoryofCropGeneticImprovement,23EcologyandPhysiology,Jin
5、an250100,China;CollegeofLifeScience,ShandongUniversity,Jinan250100,China;CollegeofAgronomy,XinjiangAgriculturalUniversity,Urumqi830000,ChinaAbstract:Diacylglycerolacyltransferase(DGAT)isarate-limitingenzymeintriacylglycerol(TAG)biosynthesispathway.Inthisst
6、udy,GenomeWalkingmethodwasusedforcloningthepromotersequenceofAhDGAT2agenefromLuhua14,andfinallya1200bpfragmentflanking5′-upstreamofAhDGAT2awasobtainedandnamedaspAhDGAT2a.ThecrucialregulatoryelementsinpAhDGAT2awerefurtheranalyzedwithsoftwarePlantCARE.Therew
7、eremanyTATA-box,CAAT-box,lightregulation,stressanddefenseresponseandhormoneresponseelements.ToassesstheactivityofpAhDGAT2a,weconstructedpAhDGAT2a:GUScassettesandintroduceditintothetobaccoSR1genomebyAgrobacterium-mediatedtransformation.Expressionpatternwasm
8、onitoredbyhistochemicalstaining.ResultsshowedthatGUSactivitydrivenbythepAhDGAT2awasdetectedinalmostallvegetativeandreproductivetissues,withahigherexpressionlevelinstigma,antherandyoungseedsthanintheotherorgan
