提高植物油营养价值和功能的基因改造(英文版)

GeneticModificationofVegetableOilsforIncreasedNutritionalValueandFunctionalityContentsoftoday’spresentation1.IntroductionofCSIROPlantIndustry2.Backgroundofplantlipidbiochemistry3.Methodologyofgenesilencing4.Oilmodificationforimprovednutritionalvalue5.OilmodificationforindustrialusesOilmodificationforneutraceuticaluses e.gomega-3fattyacidsContentsoftoday’spresentation1.IntroductionofCSIROPlantIndustry2.Backgroundofplantlipidbiochemistry3.Methodologyofgenesilencing4.Oilmodificationforimprovednutritionalvalue5.OilmodificationforindustrialusesOilmodificationforneutraceuticaluses e.gomega-3fattyacidsCSIROPlantIndustry(PI)sitesDIVISIONALHEADQUARTERSCanberraMerbein

LaboratoryAdelaideLaboratoryPerthLaboratoryAthertonLaboratoryTownsvilleBrisbaneNarrabriDarwinGinninderraExperimentStationStaffinPI116

ResearchScientists

65PostDoctoralFellows

69

Students(PhD,Honours,Masters,etc)370

ScientificSupport145 AdministrativeandSimilarSupport24 HonoraryFellows55 VisitingScientists/Contractors844

TOTALResearchEffortAppropriation55%External45%Fundingsources(PI)$78MillionResearchprogramsinPIGenomicsand

PlantDevelopmentLizDennisGeneticEngineeringforPlantImprovementJeffEllisCottonImprovementandProductionGregConstableHighPerformanceCrops

forAustraliaRichardRichardsHorticulturalCropImprovementAnnaKoltunowAustralianFloraResources&ManagementAndrewYoungHorticulturalSystemsRobWalkerMediterraneanCropsandPasturesMickPooleGeneTechnologyinCerealImprovementMatthewMorellProfitableSustainableAgricultureJohnDonnellyIntegratedCropPhysiologyandGeneticImprovementJohnManners

Cottonseedoil(CRDC)Industrialfattyacids(BASF,GRDC)Omega-3fattyacids(CSIROFlagshipProject)CanolaandB.junceaoil(MelbourneUniversity)MajorresearchactivitiesintheOilGroupContentsoftoday’spresentation1.IntroductionofCSIROPlantIndustry2.Backgroundofplantlipidbiochemistry3.Methodologyofgenesilencing4.Oilmodificationforimprovednutritionalvalue5.OilmodificationforindustrialusesOilmodificationforneutraceuticaluses e.gomega-3fattyacidsWorldproductionofvegetableoilsCottonseedOilvaluableco-productoffibre20-25%oilcontentfoodingredientcookingoilSoybean26.4Oilpalm22.8Rapeseed14.5Sunflower9.4Peanut4.7Cottonseed3.9TheBigSixVegetableOils(MT)StableUnstable16:0palmitic18:0stearic18:3linolenic18:2linoleic18:1oleicPUFAMUFALDLLDLcholesterolneutralstable&healthycookingoils26%2%15%56%<1%

15

12

9KASIISATURATESFattyacidcompositionincottonseedoilCisvs.TransfattyacidsCHHR’R”=CC=CHHRRcistransKeyenzymesinfattyacidbiosynthesis16:018:118:218:318:09desaturase12desaturase15desaturaseplastid(soluble)ERmembraneboundKASII16:018:118:218:09desaturase12desaturaseplastid(soluble)ERmembraneboundKeyenzymesinfattyacidbiosynthesisCloningoffattyacidsyntheticgenesStearoyl-ACPΔ9Desaturase(SAD)HighlydivergentinfunctionIdentifyingseed-specificgenesKeyTargetEnzymes:MicrosomalΔ12Desaturase(FAD2)Palmitoyl-ACPThioesterase(FatB)KetoAcylSynthaseII(KASII)CyclopropaneSynthaseEachenzymeisencodedbyamultigenefamilySpatially&temporallyregulatedHindIIIXbaI54321543211.G.barbadense(AD)2.G.hirsutum(AD)3.G.hirsutum(AD)4.G.herbaceum(A)5.G.raimondii(D)HindIIIXbaIΔ9-desaturaseHighlydivergentinfunctionIdentifyingseed-specificgenesKeyTargetEnzymes:MicrosomalΔ12Desaturase(FAD2)Palmitoyl-ACPThioesterase(FatB)KetoAcylSynthaseII(KASII)CyclopropaneSynthaseEachenzymeisencodedbyamultigenefamilySpatially&temporallyregulatedghFAD2-1isspecificallyexpressedindevelopingcottonembryosatmiddlematuritystage2530354045DaysAfterAnthesisΔ12-desaturaseStearoyl-ACPΔ9Desaturase(SAD)CloningoffattyacidsyntheticgenesCommonmeansofgeneticengineeringAddingnoveltraitsbyintroductionofnewgenesRemovingundesirabletraitsbygenesilencingCommonmeansofgeneticengineeringAddingnoveltraitsbyintroductionofnewgenesRemovingundesirabletraitsbygenesilencingHigh-oleicHigh-stearicLow-palmiticCocoa-buttersubstituteHydroxy-fattyacidsEpoxy-,acetylenicfattyacidsCyclopropanefattyacidsOmega-3fattyacidsContentsoftoday’spresentation1.IntroductionofCSIROPlantIndustry2.Backgroundofplantlipidbiochemistry3.Methodologyofgenesilencing4.Oilmodificationforimprovednutritionalvalue5.OilmodificationforindustrialusesOilmodificationforneutraceuticaluses e.gomega-3fattyacidsInvertedRepeat(IR)HighlyefficientSensecosuppressionAntisenseterminatorpromoterhomologousDNAtothetargetgeneLowefficiency&unpredictabilityClassicandRNAiposttranscriptionalgenesilencing(PTGS)ConstructionofRNAitransgeneInvertedrepeatofcottonfattyaciddesaturasegenewasmadeanddrivenbyaseed-specificpromoterInvertedrepeatofcottonfattyaciddesaturasegenewasmadeanddrivenbyaseed-specificpromoterLiuetal.PlantPhysiol.2002ConstructionofRNAitransgeneAAAAAAAAAAAAAATranscriptionofhair-pinRNAPTGSismediatedbydouble-strandedRNAdsRNADICERsiRNA22-25ntRISCdsRNADICERRISCAAAAAPTGSismediatedbydouble-strandedRNAInvertedrepeatofcottonfattyaciddesaturasegenewasmadeanddrivenbyaseed-specificpromoterConstructionofRNAitransgeneCodingregionInvertedrepeatofcottonfattyaciddesaturasegenewasmadeanddrivenbyaseed-specificpromoterConstructionofRNAitransgeneSpliceableintronAspliceableintronasthespacercansubstantiallyincreasetheefficacyofPTGSIntroninterruptedRNAitransgeneGTAGSpliceableintronShortDNAfragmentsuchasuntranslatedregion(UTR)wasfoundtobeeffectiveintargetinggenesspecificallyTransgenewithinvertedrepeatofUTRGTAG98-bpUTRSpliceableintronGTAG98-bpUTRStackingmultiplepairsofIRsofshortUTRscanleadtosimultaneoussilencingofmultipletargetgenesSimultaneoussilencingofmultiplegenesSpliceableintronΔ12desaturaseFAD2-1High-oleicΔ9desaturaseSAD-1High-stearicPalmitoyl-ACPthioesteraseLow-palmiticGTAG98-bpUTRSimultaneoussilencingofmultiplegenes1:untransformed2:HScotton3:HOcotton4:HS+HOhybridEmbryoRNAKASII

12Desaturase1234

9DesaturaseGene-specificRNAdegradationThetargetgenewassilencedbygene-specificRNAdegradationincottonseeds,asaconsequenceoftransformationwithhpRNAtransgene.Contentsoftoday’spresentation1.IntroductionofCSIROPlantIndustry2.Backgroundofplantlipidbiochemistry3.Methodologyofgenesilencing4.Oilmodificationforimprovednutritionalvalue5.OilmodificationforindustrialusesOilmodificationforneutraceuticaluses e.gomega-3fattyacids1525-572CottonseedOil(CSO)$18:116:018:318:218:0DevelopmentofHO-CSOforcommercialfryingfoodapplications$

12-desaturasehpRNACompletecodingregionofghFAD2-1with5’invertedrepeat(850nt)7817-41High-Oleic(HO-CSO)

DevelopmentofHO-CSOforcommercialfryingfoodapplications$

Concentrationofaldehydeinacceleratedoxidationcondition7817-41High-Oleic(HO-CSO)4.0E+07Total

AldehydesCSOHO-1HO-2OilType3.0E+072.0E+071.0E+070.0E+07$

Concentrationofaldehydeinacceleratedoxidationcondition7817-41High-Oleic(HO-CSO)4.0E+07Total

AldehydesCSOHO-1HO-2OilType3.0E+072.0E+071.0E+070.0E+07DevelopmentofHO-CSOforcommercialfryingfoodapplications$$15-3840718:116:018:318:218:0

9-desaturasehpRNAHigh-Stearic(HS-CSO)

CompletecodingregionofghSAD-1with5’invertedrepeat(580nt)DevelopmentofHS-CSOforsolid

fatapplications1525-572CottonseedOil(CSO)$18:116:018:318:218:0Cocoabutter-like16-42850

9&

12-desaturaseshpRNAs(CBS-CSO)HOandHStraitswerecombinedthroughcrossingandselection$

Developmentofcocoabuttersubstitute

cottonseedoil(CBS-CSO)1525-572CottonseedOil(CSO)18:116:018:318:218:01525-572CottonseedOil(CSO)LP+HS+HO8-41275ghFatB,ghSAD1&ghFAD2-1hpRNAsCSOTransgeniclineswithHO,HSandLPtraitswereobtainedbysimultaneoussilencingofthreegeneswithasingleIR-transgeneconstruct$Developmentoflow-palmiticcottonseedoil(LP-CSO)

$Fattyacidsynthesis16:0-ACP18:0-ACP18:1-ACPFattyacidpoolFatBKASIISAD1PLASTIDCYTOPLASMFine-tuningofcocoabutter-likeCSOTAGKEYSTRATEGY:Toincreasethepalmiticacidcontentbyeitherover-expressionofa16:0specificFatBorthedown-regulationofKASIIATTARGET:30%eachofpalmitic,stearicandoleicacidsContentsoftoday’spresentation1.IntroductionofCSIROPlantIndustry2.Backgroundofplantlipidbiochemistry3.Methodologyofgenesilencing4.Oilmodificationforimprovednutritionalvalue5.OilmodificationforindustrialusesOilmodificationforneutraceuticaluses e.gomega-3fattyacidsTransgenicproductionofindustrialFAsCrepisBalsamVernoniaCastorCCOCCOHCCCCMulti-functionalFAD2familyCCCCOΔ12ConjugaseΔ12acetylenaseΔ12EpoxygenaseCC18:218:1Δ12DesaturaseCCOHΔ12HydroxylaseLowexpressionlevelintransgenicplantsPossibleunmatchedenzymeinteractionsCyclopropane&cyclopropenefattyacidsC7CCC6CH3HOOCCDihydrosterculicacid(DHS)C6CCC6CH3HOOCCDihydromalvalicacid(DMV)C6CCC6CH3HOOCCMalvalicacid(MVL)C7CCC6CH3HOOCCSter