中期考核

扬州大学毕业设计中期考核汇报指导老师：鲁玉柱汇报人：周春艳扬州大学生物科学与技术学院生技1301班水稻miR396启动子的表达特性目录2.assignment1.background3.5.结论及其他6.未来实验计划4.问题产生的原因分析1.background•AboutmiR396:Basedonthecomputationalandexperimentalmeans,miR396wasclonedandidentifiedinbothArabidopsisandrice.ItisaveryhighlyconservedmicroRNAinplants.Itstargetgenes,GRF(GrowthRegulativeFactor),werebelievedasGA-inducedgene.However,themechanismofthisinductioninunknown.Ourpreviousworkshowedthatthetransgenicriceover-expressingmiR396presentedasadwarfphenotype.Thephenotypeofriceover-expressingmiR396•OursomepreviousresultsindicatedthatthereasonofthepositiveresponseofGRFtoGAiscausedbynegativeresponseofmiR396toGATheexpressionalpatternofagivengeneisasignificantindicatorforexploringthefunctionofthisgene.Inthisstudy,wewanttoinvestigatetheexpressionalpatternofmiR396bythemethodofdrivingtheexpressionofareportergene,GUS,bythepromotorofmiR396.PartidentifiedsequenceofthepromoterofmiR396ItisdifficulttodirectlydetecttheexpressionofmicroRNAbyNorthernblot,butitisveryeasytoobservetheexpressionalpatternofareportergene.Bytheacknowledgethatagene’sexpressionalfeatureisdeterminedbyitspromoter,thepromoterofmiR396wassuccessfullyclonedandconstructedintoexpressionalvectortodrivetheexpressionofGUS•ToinvestigatetheexpressionalpatternofGUS,whoseexpressionalpatterniscontrolledbythepromotorofmiR396•ToinvestigatewhethertheexpressionofGUSisnegativelyregulatedbyGA•ToinvestigatetheexpressionalofGUSundervariousstresses,includingbioticandabioticstresses.2.Themainpurposeofmystudy3.Someresultsofmycurrentwork•获得一个过表达miR396的转基因水稻。•12个水稻GRF（GrowthRegulatingFactor）中的11个是miR396的靶基因受miR396的负调节。我们发现，先前报道的水稻GRF基因正响应GA的原因是因为miR396负响应GA所引起的。•利用突变体slr1（水稻DELLA基因功能丧失型）和miR396的过表达突变体，还发现，miR396处于DELLA蛋白的下游。•另外，miR396过表达矮化植株的GRF和细胞周期基因处于抑制状态，施加外源赤霉素既不能恢复株高和GRF的表达，也不能恢复原响应赤霉素的细胞周期基因，这说明它们同处于一条赤霉素信号传导路径上。主要研究内容：miR396的表达分析miR396在赤霉素信号传导中的作用模式miR396在赤霉素信号传导中的作用模式当水稻中某些组织的GA含量上升时，会引起DELLA蛋白的降解，因而引起miR396表达的下降，引起GRF（靶基因）表达的上升，引起细胞周期基因表达的上升，从而引发细胞的增生，促进株高生长；反之，会抑制细胞的增生而抑制株高。研究方法•GUS染色问题产生的原因分析引出的问题：•那么miR396既然在DELLA蛋白的下游，它是否是DELLA蛋白的直接目标（或之一）？•或者DELLA蛋白通过作用于其它转录因子而间接作用于某（些）基因的启动子，如同作用于转录因子PIF4和PIF3一样（DeLucasetal.,2008;Feng.,2008）。解决方案：•克隆miR396的启动子,构建表达载体•克隆SLR1基因并在真核表达系统进行蛋白质表达；克隆并表达已知的和SLR1蛋白相互作用的转录因子（如PIF4和PIF3）•将miR396的启动子和PIF进行酵母单杂交结论及其他已取得的研究结果：1）分析MiR396启动子的成员间的序列，分析疑似DNA原件。2）克隆了系列MIR396启动子，构建了Promoter+Gus表达载体。3）克隆SLR1基因，将该基因转进MIR396启动子转基因水稻。未来实验计划1）对转Promoter植株喷施外源GA，通过GUS表达情况，分析Promoter表达。2）对于共转的转基因植株，通过GUS染色分析过表达的DELLA(SLR1)蛋白对MIR396表达的影响。3）利用酵母单杂交分析验证MIR396与光敏色素转录因子（PIF)是否存在互作关系参考文献[1]程式华（2008）.2008年中国水稻产业发展报告[M].北京：中国农业出版社，鲁玉柱，封振，边黎颖，梁建生（2009）植物发育与microRNA西北植物学报2009，29（5）：1066-1072.[2]AxtellMJ,SnyderJA,BartelDP(2007)CommonfunctionsfordiversesmallRNAsoflandplants.PlantCell.200719(6):1750-69.[3]Cao,D.etal.(2006)GibberellinmobilizesdistinctDELLA-dependenttranscriptomestoregulateseedgerminationandfloraldevelopmentinArabidopsis.PlantPhysiol.142,509–525[4]Carrera,E.,Bou,J.,Garcia-Martinez,J.L.,andPrat,S.（2000）ChangesinGA20-oxidasegeneexpressionstronglyaffectstemlength,tuberinductionandtuberyieldofpotatoplants,PlantJ.,22,247–256.[5]ChoiD,KimJH,KendeH.(2004)WholegenomeanalysisoftheOsGRFgenefamilyencodingplant-specificputativetranscriptionactivatorsinrice(OryzasativaL.).PlantCellPhysiol.45(7):897-904.[6]Coles,J.P.,Phillips,A.L.,Croker,J.,Garcia-Lepe,R.,Lewis,M.J.,andHedden,P.（1999）ModificationofgibberellinproductionandplantdevelopmentinArabidopsisbysenseandantisenseexpressionofgibberellin20-oxidasegenes,PlantJ.,17,547–556.参考文献[7]Curtis,I.S.,Ward,D.A.,Thomas,S.G.etal.（2000）InductionofdwarfismintransgenicSolanumdulcamarabyoverexpressionofagibberellin20-oxidasecDNAfrompumpkin,PlantJ.,23,329–338.[8]DeLucasM,DavièreJM,Rodríguez-FalcónM,PontinM,Iglesias-PedrazJM,LorrainS,FankhauserC,BlázquezMA,TitarenkoE,PratS.(2008)Amolecularframeworkforlightandgibberellinscontrolofcellelongation.Nature451,480–484[9]FengS,MartinezC,GusmaroliG,WangY,ZhouJ,WangF,ChenL,YuL,Iglesias-PedrazJM,KircherS,SchäferE,FuX,FanLM,DengXW.(2008)CoordinatedregulationofArabidopsisdevelopmentbylightandplanthormonegibberellins.Nature451,475–479[10]GaoP,BaiX,YangL,LvD,LiY,CaiH,JiW,GuoD,ZhuY.（2010）Over-expressionofosa-MIR396cdecreasessaltandalkalistresstolerance.Planta.231(5):991-1001.[11]HiranoK,Ueguchi-TanakaM,MatsuokaM.(2008)GID1-mediatedgibberellinsignalinginplants.TrendsPlantSci.13(4):192-9[12]Ikeda,A.(2001).slenderrice,aconstitutivegibberellinresponsemutant,iscausedbyanullmutationoftheSLR1gene,anorthologoftheheight-regulatinggeneGAI/RGA/RHT/D8.PlantCell13,999–-1010参考文献[13]Itoh,H.,Ueguchi-Tanaka,M.,Sato,Y.,Ashikari,M.&Matsuoka,M.(2002)ThegibberellinsignallingpathwayisregulatedbytheappearanceanddisappearanceofSLENDERRICE1innuclei.PlantCell14,57–-70.[14]Jones-RhoadesMW,BartelDP(2004).Computationalidentificationofplantmicro-RNAsandtheirtargets,includingastress-inducedmiRNA.Mol.Cell14,787–99.[15]Jones-rhoadesMW,BartelDP,BartelB(2006),MicroRNAsandtheirregulatoryrolesinplants.AnnuRevPlantBiol57,19-53.[16]KimJH,ChoiD,KendeH.(2003)TheAtGRFfamilyofputativetranscriptionfactorsisinvolvedi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