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EnhancedADP-glucosepyrophosphorylaseactivityinwheatendospermincreasesseedyield*DepartmentofPlantSciences,MontanaStateUniversity,Bozeman,MT59717;and†HorticulturalSciencesDepartment1143FifieldHall,UniversityofFlorida,Gainesville,FL32611outline•Abstract•Introduction•MaterialsandMethods•Results•Discussion•目的•Yieldincerealsisafunctionofseednumberandweight;bothparametersarelargelycontrolledbyseedsinkstrength.TheallostericenzymeADP-glucosepyrophosphorylase(AGP)playsakeyroleinregulatingstarchbiosynthesisincerealseedsandislikelythemostimportantdeterminantofseedsinkstrength.Abstract•方法•PlantAGPsareheterotetrameric,consistingoftwolargeandtwosmallsubunits.Wetransformedwheat(TriticumaestivumL.)withamodifiedformofthemaize(ZeamaysL.)Shrunken2gene(Sh2r6hs),whichencodesanalteredAGPlargesubunit.•ThealteredlargesubunitgivesrisetoamaizeAGPheterotetramerwithdecreasedsensitivitytoitsnegativeallostericeffector,orthophosphate,andmorestableinteractionsbetweenlargeandsmallsubunits..•结果：•TheSh2r6hstransgenewasstillfunctionalafterfivegenerationsinwheat.DevelopingseedsfromSh2r6hstransgenicwheatexhibitedincreasedAGPactivityinthepresenceofarangeoforthophosphateconcentrationsinvitro.•TransgenicSh2r6hswheatlinesproducedonaverage38%moreseedweightperplant.Totalplantbiomasswasincreasedby31%inSh2r6hsplants•结论•Resultsindicateincreasedavailabilityandutilizationofresourcesinresponsetoenhancedseedsinkstrength,increasingseedyield,andtotalplantbiomass.Introduction•Starchisthemajorcomponentofwheatyield,comprising70%ofwheatseeddryweight.•ADP-glucosepyrophosphorylase(AGP),anallostericallyregulatedheterotetramerconsistingoftwolargeandtwosmallsubunits,catalyzestherate-limitingreactioninstarchbiosynthesisinplants.•作用机理•AGPusesthesubstratesglucose1-phosphateandATPtoproduceADPglucoseandpyrophosphate.•ADP-glucoseisthenusedastheglucosedonorforstarchsynthases.•ThepositiveallostericeffectorofAGPis3-phosphoglycerate,whereasthenegativeallostericeffectorisorthophosphate(Pi).•MaizeendospermAGPlargesubunitsareencodedbyShrunken2(Sh2),whereassmallsubunitsareencodedbyBrittle2(Bt2).•InhibitionofAGPactivitybyPiappearstolimitstarchbiosynthesisandyieldincropplants.•EvidenceforthishascomefromstudiesofamodifiedAGPlargesubunitinmaizeandfromuseofanalteredbacterialAGPinpotato•AGPisperhapsthemostheat-labilestarchbiosyntheticenzymeinmaize.•Asingleaminoacidsubstitutioninthemaizelargesubunitthatconditionsmorestablelargesubunit–smallsubunitinteractionsmayleadtoenhancedAGPactivityandincreasedyield.•本实验的目的：•HerewetestthehypothesisthatincreasedAGPactivitywithindevelopingwheatseedendospermincreasesyield.•WheatwastransformedwithamodifiedmaizeSh2gene(Sh2r6hs),whichencodedanalteredAGPlargesubunit,givingrisetoanAGPheterotetramerwithdecreasedsensitivitytoinhibitionbyPiandmorestablelargesubunit–smallsubunitinteractions.Wheatexpressingthetransgeneproducedmoreseedweightandtotalplantbiomass.•Possibleexplanationsforthisincreasedavailabilityandutilizationofresourcesarediscussed.MaterialsandMethods•PlasmidConstructsUsedforTransformation•WheatTransformation•PCRScreeningofTransgenicLines.•Southern,Northern,andWesternBlotAnalyses.•AGPActivityAssays•PlantYieldDataCollectionandAnalysis.•GreenhouseConditionsPlasmidConstructsUsedforTransformationCaMV35Snopalinesynthase1,557-bpcDNAtranscriptionstartsitetranslationinitiationsitepRQ101A•Therev6mutationisa6-bpinsertionresultingintheadditionoftyrosine（酪氨酸)andserine（丝氨酸）ataminoacidpositions495and496oftheAGPlargesubunit.ThisinsertionrendersAGPlesssensitivetoPiinhibition.•Thesecondchange,hs33,isapointmutationcausingsubstitutionataminoacidposition333oftyrosineforhistidine（组氨酸）andconfersmorestablelargesubunit–smallsubunitinteractionsintheAGPheterotetramerWheatTransformation•Immatureembryos0.5–1.5mmlongwereisolatedfromgreenhouse-grownwheatvarietyHi-LineandplacedonS1callusinductionmediumfor5–8daysinthedarkat25°C.•CalliwerethenmovedtoS1mediumsupplementedwith0.4Msorbitol4hbeforebombardment•ConstructspSh2r6hsandpRQ101Awereprecipitatedon1-um-diametergoldparticlesina1:1molarratiobyusingastandardprotocol.Calliwerebombardedtwicebyusing1,550-psirupturedisksand6-cmtargetdistanceinaBiolisticPDS-1000HeParticleDeliverySystem•After17h,calliweremovedtoaselectionmediumconsistingofS1mediumsupplemented.•Onceshootsreached0.5cminheight,plantletsweremovedtorootingmediuminthelightat25°C.•Regeneratedplantletsweretransferredtosoil,allowedtogrowfor1–3weeks,andsprayedwith0.1%glufosinatetoidentifytransgenics.PCRScreeningofTransgenicLines•PCRusinggenomicDNAfromT0plantswasdonetoidentifytransgeniclinescontainingtheSh2r6hssequence.•Thisprimerpairproducesanamplifiedproductof826bpSouthern,Northern,andWesternBlotAnalyses•Southern•20ugofgenomicDNAisolatedfromhomozygousT2plantswasdigestedwithEcoRI,fractionatedon0.7%agarosegels,andtransferredtoanylonmembrane•Themembranewasprobedwiththe32P-labeledSh2r6hscodingsequencelabeledbytherandomprimermethod•Northern•RNAforNorthernblotanalysiswasextractedfrom20dayspostanthesis(dpa)homozygousT3seedsgroundinliquidnitrogen,byusingaLiClmethod•TenmicrogramsoftotalRNApe