新型棉籽油–應用RNAi改良脂肪酸代謝途徑

CSIRO.GeneticmodificationofCottonseedOilCotton–adualpurposecropSoybean38Oilpalm41Rapeseed18Sunflower10Peanut5Cottonseed5CottonseedOilvaluableco-productoffiberfoodingredientcookingoilwideapplicationsinfoodindustry(Millionmetrictons)CSIRO.GeneticmodificationofCottonseedOilGeneticimprovementofcottonseedoilHighstabilityoilforthefoodindustry–HigholeicHighvaluespecialityfoodfat-CocoabuttersubstituteNutraceuticalapplication-Omega-3vegetableoilsIndustrialfattyacids-Biodiedel&industrialfeedstockCSIRO.GeneticmodificationofCottonseedOilGeneticimprovementofcottonseedoilHighstabilityoilforthefoodindustry–HigholeicHighvaluespecialityfoodfat-CocoabuttersubstituteNutraceuticalapplication-Omega-3vegetableoilsIndustrialfattyacids-Biodiedel&industrialfeedstockCSIRO.GeneticmodificationofCottonseedOilFattyacidcompositionof

oilseedcropsMajoroilseedcropslimitednumberoffattyacidsHighlyvariableFAcontentCottonseedoil(CSO)highinsaturatehighinpolyunsaturatedFACSIRO.GeneticmodificationofCottonseedOilNomenclatureoffattyacidsStearicacid(18:0)Oleicacid(18:1)Linoleicacid(18:2)Palmiticacid(16:0)OOHOOHOOHOOHOOHω-6ω-3DHA(22:6)CSIRO.GeneticmodificationofCottonseedOilStructureandpropertyoffattyacidsOleicacid(18:1)Stearicacid(18:0)Linolenicacid(18:3)DHA(22:6)StableVeryUnstableLDL-raisingLDL-loweringLDL-neutralStableStableLDL-loweringMp.63Mp.-55Mp.70Mp.16Palmiticacid(16:0)UnstableLDL-loweringMp.-11CSIRO.GeneticmodificationofCottonseedOilWhatisatransfattyacid?TransfattyacidisanunsaturatedfattyacidwithadoublebondintransconfigurationStearicacid(C18:0)eladicacid(C18:1trans)oleicacid(C18:1cis)CSIRO.GeneticmodificationofCottonseedOilTransfattyacidiscontroversial….Partialhydrogenationhasbeenwidelyusedtoincreaseoxidativestability,extendshelflife,andcreateplasticityforsolidfatapplicationsHydrogenatedvegetableoilswasonceregardedasahealthieralternativetosaturatedfattyacids.Itiswidelyusedinthefoodindustry.Scientificresearchindicatedtransfattyacidscouldbeanimportant

causeofCHD.Itisestimatedtransfattyacidscaused30,000/yearCHDdeathintheUSACSIRO.GeneticmodificationofCottonseedOilUndesirablehealtheffectsoftransfatCoronaryheartdiseaseHumanlipaseenzymeisspecifictocis,nottotransNurses’HealthStudy(NHS)with120,000femalenursesForeach2%increaseintransFAindiet,coulddoubletheCHDrisk.Thesameeffectcouldbeachievedby15%increaseofsaturatedFA.Othereffects(withoutscientificconsensus)Alzheimer’sdiseaseCancerDiabetesObesityLiverdysfunctionInfertilityCSIRO.GeneticmodificationofCottonseedOilTransfattyacidisunderthespotlightCSIRO.GeneticmodificationofCottonseedOilGovernmentregulationsabouttransfatintheworldUSArequestedtranslabellingsince2006.NewYorkcityisthefirstlargeUScitytobantransinrestaurant.OnJuly25,2008,CaliforniahasbecomethefirstUSstatetobantransfatinrestaurant.ItislikelymanyothercountrieswillfollowthetrendofbanningtransfatconsumptionDenmarkwasthefirstcountrytointroducelaweffectivelybanspartiallyhydrogenatedoils,since2003.ThiswasfollowedbySwitzerland.CSIRO.GeneticmodificationofCottonseedOilExcellentopportunityforHOoilsHigh-oleicoilsareoxidativelystable,butalsogoodforcholesterolmetabolism.High-oleicoilsdoesnotrequirehydrogenation,thereforenotransfattyacidsDuPont/BungeLimited

-HOsoybeanwillbecommercialisedin2009CSIRO

-HOcotton/MonoCottcottonCSIRO.GeneticmodificationofCottonseedOilLipidbiosynthesisindevelopingseedsERAcetyl-CoAKASIISADPlastidKASIIIKASIG3PLPAPADAGTAGGPATLPATPAPDGATFAD2FAD3LPCATFatAFatBFatBLPCAT,PLACPT,PDATC18:2PCC2:0ACPC4:0ACPC16:0ACPC18:0ACPC18:1ACPCoAC16:0CoAC18:0CoAC18:1FattyacidCoAC18:3PCC18:1PCCSIRO.GeneticmodificationofCottonseedOilLipidbiosynthesisindevelopingseedsERKASIISADPlastidFAD2FatBC18:2PCC16:0ACPC18:0ACPC18:1ACPCoAC16:0CoAC18:1C18:1PCTAG26%15%CoAC18:02%58%

CSIRO.GeneticmodificationofCottonseedOilLipidbiosynthesisindevelopingseedsERKASIISADPlastidFAD2FatBC18:2PCC16:0ACPC18:0ACPC18:1ACPCoAC16:0CoAC18:1C18:1PCTAG26%CoAC18:02%

4%80%CSIRO.GeneticmodificationofCottonseedOilLipidbiosynthesisindevelopingseedsERKASIISADPlastidFAD2FatBC18:2PCC16:0ACPC18:0ACPC18:1ACPCoAC16:0CoAC18:1C18:1PCTAG26%CoAC18:02%

4%80%C19:0PCC19:1PCC18:1PCCPADCPEOCPASCPFACSIRO.GeneticmodificationofCottonseedOilLipidbiosynthesisindevelopingseedsERKASIISADPlastidFAD2FatBC18:2PCC16:0ACPC18:0ACPC18:1ACPCoAC16:0CoAC18:1C18:1PCTAG26%CoAC18:02%

4%80%C19:0PCC19:1PCC18:1PCCPADCPEOCPASCPFA

CSIRO.GeneticmodificationofCottonseedOilLipidbiosynthesisindevelopingseedsERKASIISADPlastidFAD2FatBC18:2PCC16:0ACPC18:0ACPC18:1ACPCoAC16:0CoAC18:1C18:1PCTAG26%CoAC18:02%

4%80%C19:0PCC19:1PCC18:1PCCPADCPEOCPASCPFA

CSIRO.GeneticmodificationofCottonseedOilLipidbiosynthesisindevelopingseedsERKASIISADPlastidFAD2FatBC18:2PCC16:0ACPC18:0ACPC18:1ACPCoAC16:0CoAC18:1C18:1PCTAG26%CoAC18:02%

4%80%C19:0PCC19:1PCC18:1PCCPADCPEOCPASCPFA

CSIRO.GeneticmodificationofCottonseedOilLipidbiosynthesisindevelopingseedsERKASIISADPlastidFAD2FatBC18:2PCC16:0ACPC18:0ACPC18:1ACPCoAC16:0CoAC18:1C18:1PCTAG7%CoAC18:02%

4%80%C19:0PCC19:1PCC18:1PCCPADCPEOCPASCPFA

CSIRO.GeneticmodificationofCottonseedOilHowtomakeanRNAitransgeneAtypicaltransgenecontainspromoter,terminatorandtargetgenesequencesPromoterTerminatorTargetgeneCSIRO.GeneticmodificationofCottonseedOilInvertedrepeatofcottonfattyaciddesaturasegenewasmadeanddrivenbyaseed-specificpromoterHowtomakeanRNAitransgeneCSIRO.GeneticmodificationofCottonseedOilAAAAAAAAAAAAAAHowRNAiworks?CSIRO.GeneticmodificationofCottonseedOildsRNADICERsiRNA22-25ntRISCHowRNAiworks?CSIRO.GeneticmodificationofCottonseedOildsRNADICERRISCAAAAAHowRNAiworks?CSIRO.GeneticmodificationofCottonseedOildsRNADICERRISCHowRNAiworks?CSIRO.GeneticmodificationofCottonseedOilInvertedrepeatofcottonfattyaciddesaturasegenewasmadeanddrivenbyaseed-specificpromoterRNAi干擾基因構建CSIRO.GeneticmodificationofCottonseedOilCodingregionInvertedrepeatofcottonfattyaciddesaturasegenewasmadeanddrivenbyaseed-specificpromoterRNAi干擾基因構建CSIRO.GeneticmodificationofCottonseedOilSpliceableintronAspliceableintronasthespacercansubstantiallyincreasetheefficacyofPTGS含intron的RNAi基因構建GTAGCSIRO.GeneticmodificationofCottonseedOilSpliceableintronShortDNAfragmentsuchasuntranslatedregion(UTR)wasfoundtobeeffectiveintargetinggenesspecificallyUTR反向重復序列-增加基因特異性GTAG98-bpUTRCSIRO.GeneticmodificationofCottonseedOilSpliceableintronGTAGStackingmultiplepairsofIRsofshortUTRscanleadtosimultaneoussilencingofmultipletargetgenes一石多鳥-多基因的同步沉默CSIRO.GeneticmodificationofCottonseedOilSpliceableintronΔ12desaturaseFAD2-1High-oleicCyclopropaneFAsynthaseLow-CPFAsPalmitoyl-ACPthioesteraseLow-palmiticGTAG一石多鳥-多基因的同步沉默CSIRO.GeneticmodificationofCottonseedOilSimultaneoustargetingofthreegenesbyRNAiTG1:ghFAD2-1TG2:eitherghCPA-FAS2TG3:ghFatB-2TermNOSTermPromNPTIISelectablemarkerLec-TLec-PspacerRNAisilencingconstructTG2TG1TG3CSIRO.GeneticmodificationofCottonseedOil15251572CottonseedOil18:116:0CPFA18:218:07817241High-OleicHigh-oleic&MonoCottcottonseedoils8070.2101MonoCottFAD2

FAD2/FatB/CPA-FAS

CSIRO.GeneticmodificationofCottonseedOilConcentrationofaldehydeinacceleratedoxidationcondition4.0E+07TotalAldehydesCSOHO-1HO-2OilType3.0E+072.0E+071.0E+070.0E+07High-oleicoilishighlystable,nohydrogenationisrequiredCSIRO.GeneticmodificationofCottonseedOilPalmitic/oleicratioinMonoCottCoker315controlT2

T4

CSIRO.GeneticmodificationofCottonseedOilLowCPFAinMonoCottCSIRO.GeneticmodificationofCottonseedOilGeneticimprovementofcottonseedoilHighstabilityoilforthefoodindustry-HigholeicHighvaluespecialityfoodfat-CocoabuttersubstituteNutraceuticalapplication-Omega-3vegetableoilsIndustrialfattyacids-Biodiedel&industrialfeedstockCSIRO.GeneticmodificationofCottonseedOilCocoabutter–themosthighlypricedplantlipidCSIRO.GeneticmodificationofCottonseedOilWhatisspecialaboutcocoabutterThemeltingpointCocoabutter 35°CCottonseedoil 4°CThemeltingcurveCocoabutter: Sharp

Cottonseedoil: gradientThefattyacids

Palmitic Stearic OleicLinoleicCocoabutter 26 3236 -Cottonseedoil 26 2 15 58TheTAGstructureCocoabutter: POS,POP,SOS

Cottonseedoil: PLL,LOP

CSIRO.GeneticmodificationofCottonseedOilLipidbiosynthesisindevelopingseedsERKASIISADPlastidFAD2FatBC18:2PCC16:0ACPC18:0ACPC18:1ACPCoAC16:0CoAC18:1C18:1PCTAG26%15%CoAC18:02%58%

CSIRO.GeneticmodificationofCottonseedOilLipidbiosynthesisindevelopingseedsERKASIISADPlastidFAD2FatBC18:2PCC16:0ACPC18:0ACPC18:1ACPCoAC16:0CoAC18:1C18:1PCTAG16%15%CoAC18:038%38%

CSIRO.GeneticmodificationofCottonseedOilCombinationofHO&HStraitsPalmiticStearicOleicLinoleicClassiccottonHOcottonHScotton26215581617851625-40735F2F3CSIRO.GeneticmodificationofCottonseedOilTheHScottonhasrelativelyhighmeltingpointat15-20°C,andthemeltingcurveisrelativelysharp.Meltingcurveofhigh-steariccottonseedoilCSIRO.GeneticmodificationofCottonseedOilLipidbiosynthesisindevelopingseedsERKASIISADPlastidFAD2FatBC18:2PCC16:0ACPC18:0ACPC18:1ACPCoAC16:0CoAC18:1C18:1PCTAG26%15%CoAC18:058%

2%CSIRO.GeneticmodificationofCottonseedOilLipidbiosynthesisindevelopingseedsERKASIISADPlastidFAD2FatBC18:2PCC16:0ACPC18:0ACPC18:1ACPCoAC16:0CoAC18:1C18:1PCTAG40-75%10%CoAC18:01%18%

CSIRO.GeneticmodificationofCottonseedOilKIR-10:ModerateincreaseofpalmiticacidSampleC14:0C16:0C16:1C16:2C18:0C18:1(n-9)C18:1(n-7)C18:2C18:3C20:0KIR-10_230.248.09.41.30.93.71.534.30.10.2KIR-10_20.622.30.50.01.813.30.860.10.10.2CSIRO.GeneticmodificationofCottonseedOilKIR-1:veryhighpalmiticacidC14:0C16:0C16:1C16:2C18:0C18:1n-9C18:1n-7C18:2C18:3C20:0KIR-1_130.725.80.50.02.211.10.758.50.10.3KIR-1_140.178.75.71.61.30.70.810.20.00.4CSIRO.GeneticmodificationofCottonseedOilGeneticimprovementofcottonseedoilHighstabilityoilforthefoodindustry-HigholeicHighvaluespecialityfoodfat-CocoabuttersubstituteNutraceuticalapplication-Omega-3vegetableoilsIndustrialfattyacids-Biodiedel&industrialfeedstockCSIRO.GeneticmodificationofCottonseedOilOmega-3functionalfoodCSIRO.GeneticmodificationofCottonseedOil18:118:016:020:420:322:522:4w-6pathwayg-18:3?6-des?5-elo?5-des?6-elo20:518:420:422:6w-3pathway22:5Mammals24:524:6b-oxidation?7-elo?6-Des24:424:5b-oxidation?7-elo?6-Des18:2a-18:3EssentialFAFromdietFattyacidbiosynthesisinmammalsCSIRO.GeneticmodificationofCottonseedOil18:118:016:020:420:322:522:4w-6pathwayg-18:320:518:420:422:6w-3pathway22:5Mammals24:524:6b-oxidation?7-elo?6-Des24:424:5b-oxidation?7-elo?6-Des18:2a-18:3EssentialFAFromdietFattyacidbiosynthesisinmammalsDHAEPACSIRO.GeneticmodificationofCottonseedOilSourceofomega-3longchainpolyunsaturatedfattyacidsCSIRO.GeneticmodificationofCottonseedOil?5-elo?4-des?5-des?6-elo18:118:018:216:0Fattyacidbiosynthesisinplants,fungiandalgaew-6pathwayw-3pathway20:420:322:522:418:4DHAEPASDA20:520:422:622:5Cottonseedoil?6-desa-18:3g-18:3FlaxBorage?15-desCSIRO.GeneticmodificationofCottonseedOilTransgenicre-assemblyoftheentireLC-PUFApathwayinoilseedcropsGenesPt?6DesPt?5DesPp?6EloAbbadietal(2004)Kinneyetal(2004)Robertetal(2005)Wuetal(2005)Sd?6DesMa?6DesMa?5DesSa?4DesSd?17DesMa?6EloPav?5EloDr?5/6DesCeEloPs?5EloPs?4DesPi?6DesTh?5DesTh?4DesCo?12DesPin?17DesOmEloPp?6EloTh?6EloThLPAATHostplantflaxsoybeanArabidopsis

BrassicajunceaAAEPADHA25151.51.21.50.5-19.62-3.31.51.0-CSIRO.GeneticmodificationofCottonseedOilGeneticimprovementofcottonseedoilHighstabilityoilforthefoodindustry-HigholeicHighvaluespecialityfoodfat-CocoabuttersubstituteNutraceuticalapplication-Omega-3vegetableoilsIndustrialfattyacids-Biodiedel&industrialfeedstockCSIRO.GeneticmodificationofCottonseedOilAllgoodreasonsforbiodieselCSIRO.GeneticmodificationofCottonseedOilThechoiceisobviousCSIRO.GeneticmodificationofCottonseedOilVegetableoil–theoriginaldiesel"Theuseofvegetableoilsforenginefuelsmayseeminsignificanttoday.Butsuchoilsmaybecomeinthecourseoftimeasimportantasthepetroleumandcoaltarproductsofthepresenttime"

-RudolphDiesel,1912CSIRO.GeneticmodificationofCottonseedOilSoybeanoilvsmineraloil–recentpricetrends?1973-1986MiddleEastpoliticalinstabilityseverelyaffectssupplyoutlook2004-2008HeavydemandandPeakOilspeculationincreasespriceRenewedinterestinindustrialuseofvegetableoils.Developmentofbiodieseltechnology.Economicallyviableonlywithsubsidysupport.Increasedsubstitutabilityofoils.3-yrrollingmean;1MTSBO=1111L=7x159LbarrelsCSIRO.GeneticmodificationofCottonseedOilAlternativesourceofbiodieselUsingaspeciesofalgaewithupto50%oilcontent,only0.3%ofthelandareaoftheUScouldbeutilizedtoproduceenoughbiodieseltoreplacealltransportationfueltheUSAcurrentlyutilizes.Indiangovernmenthasidentified98millionacresoflandwhereJatrophacanbegrown,hopingitwillreplace20%ofdieselconsumptioninfiveyears.CSIRO.GeneticmodificationofCottonseedOilDemandforliquidfuelcannotbemetbyplantoil3.4BTfuelsMineraloil(gasoline,diesel,etc)130MTVegetableoilsCSIRO.GeneticmodificationofCottonseedOil…butchemicalsupplyoptionsarelimited320MTpetrochemical110MTfooduse20MTindustrialuseDoesitmakesensetoassemblecomplexcarbonmoleculesinplantsandthenburnthemforenergy?Plantoilscanmakeaninterimcontributiontofuelneedsthroughbiodieseluntilhighervaluespecialtyoleochemicaltypesaredeveloped.CSIRO.GeneticmodificationofCottonseedOilEngineeringsynthesisofunusualfattyacidsPalmitic(16:0)Stearic(18:0)Oleic(18:1)Linoleic(18:2)Linolenic(18:3)CSIRO.GeneticmodificationofCottonseedOilFunctionalitiesofdivergentFad2genefamilyCCCCOΔ12ConjugaseΔ12acetylenaseΔ12EpoxygenaseCC18:218:1Δ12DesaturaseCCOHΔ12HydroxylaseCSIRO.GeneticmodificationofCottonseedOil020406080100%UFAinseedoil12C5Aleuritesfordii(tung)EleostearicCCCCCC8CrepisalpinaCrepenynicCCCCCC8C5Ricinuscommunis(castorbean)RicinoleicCC5CCCCC8OHCrepispalaestinaVernolicCCCCCC8C5OMetabolicengineeringinER:

PooraccumulationofUFAintransgenicseedoilsFad2CSIRO.GeneticmodificationofCottonseedOilLipidbiosynthesisindevelopingseedsERAcetyl-CoAKASIISADPlastidKASIIIKA