玉米粉质胚乳突变体 优质特选

玉米粉质胚乳突变体的研究,宋任涛上海大学,1,精编材料,玉米是重要的作物和优秀的研究模式,2,精编材料,WalbotandEvans(2003)NatureReviewsGenetics,4:369-379,玉米籽粒是“胚乳型”种子的代表,3,精编材料,玉米籽粒关键发育时期,SabelliandLarkins,PlantPhysiol149(2009)14-26,4,精编材料,淀粉粒(S)和蛋白体(P)是主要的储藏形式,籽粒是玉米主要的储藏器官,S,S,P,胚乳是主要的储藏组织,5,精编材料,蛋白体（PB）由内质网（ER）形成,HermanandLarkins(1999)PlantCell,11:601-613,6,精编材料,醇溶蛋白有序地包装在蛋白体中,AdaptedfromLendingandLarkins(1989)PlantCell,7,精编材料,玉米粉质胚乳突变体,8,精编材料,是早年重要的遗传突变体,FirstreportbySingletonWRandJonesDFintheearlyoflastcentury,includingopaqueendosperm1andopaqueendosperm2.EmersonRA,BeadleGWandFraserAC(1935)Asummaryoflinkagestudiesinmaize.CornellUnivAgricExpStnMemoir180:1-83,described40classicgeneticloci.,9,精编材料,粉质胚乳突变体与高赖氨酸玉米,Mertz,ETetal.(1964)Mutantgenethatchangesproteincompositionandincreaseslysinecontentofmaizeendosperm.Science145:279-280(o2).Nelson,OEJretal.(1965)Secondmutantgeneaffectingtheaminoacidpatternofmaizeendospermproteins.Science150:1469-1470(floury2(fl2),o1ando2).Misra,PSetal.(1972)Endospermproteinsynthesisinmaizemutantswithincreasedlysinecontent.Science176:1425-1426(bt2,fl2,o2ando7).,10,精编材料,玉米已知的粉质胚乳突变体,Recessiveopaquemutants(o1,o2,o5,o7,o9o11,o13o16),Semi-dominantflourymutants(fl1,fl2andfl3)Dominantmutants(McandDe-B30),BryanC.GibbonandBrianA.Larkins(2005)TrendsGenet21,227-233.,11,精编材料,已经克隆的玉米粉质胚乳突变体,12,精编材料,opaque7和opaque6,13,精编材料,opaque7(o7)突变体,14,精编材料,著名的高赖氨酸突变体,McWhirter,KS(1971)Aflouryendosperm,highlysinelocusonchromosome10.MNL45:184Misra,PSetal.(1972)Endospermproteinsynthesisinmaizemutantswithincreasedlysinecontent.Science176:1425-1426Burr,FAandBurr,B(1982)ThreemutationsinZeamaysaffectingzeinaccumulation:acomparisonofzeinpolypeptides,invitrosynthesisandprocessing,mRNAlevels,andgenomicorganization.JCellBiol94:201-206(o2,fl2,o7),15,精编材料,突变体种子蛋白普遍下降,zeins33.4%,non-zeins15.1%,totalproteins20.7%,16,精编材料,透射电镜（TEM）分析,o7,wt,2500 x,6000 x,21DAP,17,精编材料,蛋白体的统计分析,蛋白体变少变小,18,精编材料,对O7基因的图位克隆,Gene2appearedtobethecandidategeneforo7.,28kb,19,精编材料,转基因功能互作验证,TheclonedgeneistheO7gene.,20,精编材料,转基因RNAi分析,SuppressionofO7genefunctionbyRNAireproducedtheopaqueendospermphenotype.,21,精编材料,Acyl-CoASynthetase(ACS)superfamilyAcylActivatingEnzyme(AAE)family,O7编码酰基活化酶3（AAE3）基因,22,精编材料,SubstratescreeningforpurifiedGST-O7.,O7是Oxalyl-CoASynthetase,草酸,23,精编材料,A,B,O7蛋白分布在细胞质中,CFP-PTS1,GFP-O7,24,精编材料,O7在籽粒中高度表达,Nosignificantdifferenceattranscriptlevelbetweenmutantandwildtype.,25,精编材料,突变造成O7蛋白显著减少,The12-bpdeletionino7mutantallelecausedsignificantreductionofo7proteinindevelopingseeds.,26,精编材料,O7突变影响氨基酸生物合成主要路径,up-regulated,down-regulated,unchanged,27,精编材料,O7突变在水稻中同样有效,CKNTGNTGNTGTGTGTG,28,精编材料,opauqe6(proline1)突变体,29,精编材料,籽粒主要储藏成分均显著下降,Thecontentoftotalprotein,zein,starchandfatofwtandmutant,30,精编材料,o6mutantendospermhasgreatlyreducedstorageorganelles(proteinbodiesandstarchgranules),淀粉粒和蛋白体显著变小,31,精编材料,O6基因的图位克隆,7个等位突变体确认基因,32,精编材料,O6编码P5CS21-Pyrroline-5-carboxylatesynthetase,33,精编材料,SzabadosandSavour.(2009)TrandsinPlantscience,负责胞质中的脯氨酸合成,x,34,精编材料,O6主要分布在细胞质中,ThesubcellularlocalizationofO6protein,OnionCells,TobaccoCells,35,精编材料,O6具有P5CS酶活,TheenzymeactivityanalysisofrecombinantO6protein,36,精编材料,O6突变影响脯氨酸生物合成,Thefreeandtotalprolinecontentofwtandmutantkernel,37,精编材料,脯氨酸缺乏不影响储藏蛋白转录水平,Theexpressionanalysisofzeinsinwtandmutantendosperm,38,精编材料,但储藏蛋白翻译水平严重下降,Theexpressionanalysisofzeinsinwtandmutantendosperm,39,精编材料,胚乳蛋白整体翻译水平下降,Thetotalproteinanalysisofwtandpro1mutantendospermofdifferentallele,40,精编材料,翻译关键调控因子eIF2磷酸化调控,突变体eIF2磷酸化水平急剧上升，造成翻译抑制,41,精编材料,O6突变表型的形成机制,P5CS,Proline,X,UnchargedtRNApro,Translationalstress,eIF2phosphorylation,Proteinsynthesis,42,精编材料,Zein基因,Zein蛋白,蛋白体,结构基因fl2,Mc1,DeB30,转录层面o2（转录因子）,翻译层面o7,o6（氨基酸供给）,o1（蛋白体形成）,fl1（蛋白体装配）,粉质胚乳突变体与储藏蛋白调控,储运层面,43,精编材料,致谢,MaizeGeneticsStockCenterBrookhavenNationalLaboratoryDr.FrancesBurrChinaAgriculturalUniversityDr.JinshengLaiInstituteofGeneticsandDevelopmentalBiology,CASDr.LihuangZhuUniversityofArizonaDr.BrianLarkins,ShanghaiUniversityDr.GangWangD