类黄酮代谢工程报告

MetabolicEngineeringofFlavonoidBiosynthesisinApplebyGeneticTransformationHouhuaLiApple•Oneofthemostimportantfruitsoftheworld•Applesareaffectedbyalargenumberofdiseases,forexampleapplescabandfireblightFlavonoids•Polyphenoliccompounds•BasicC6-C3-C6structureOABCBasicstructureofflavonoids•Fortheplants--Plantpigments--Defenseagainstdiseases--Auxintransportinhibitors--UV-protection--Antioxidantproperties•Forthehumanhealth--Antioxidant--Anti-cancer--Influenceonbloodglucose--EnzymeinhibitionFunctionsofflavonoidsQuercetin4-Cumaroyl-CoAKaffeoyl-CoAPentahydroxychalkonEriodictyolPentahydroxy-flavanonLuteolinDihydro-myricetinLeucodelphinidinLeucocyanidinANSDFRDelphinidinNaringeninTetrahydroxychalkonTricetinApigeninCHSCHIF3‘,5‘HF3‘HFNSFHTF3‘HMyricetinKämpferolDihydro-quercetinDihydro-kaempferolF3‘,5‘HFLSLeucopelargonidinPelargonidinCyanidinFlavonoleFlavoneFlavanoneChalkoneAuroneOHOCoAsOHOHOCoAs3*Malonyl-CoAOHOOHOHOHOHOOHOHOHOHHOOOOHOHOHOHHOOOOHOHHOOOOHOHOHAnthocyanidineDihydro-flavonoleHOOOOHOHOHOHHOOOOHOHHOOOOHOHOHHOOOOHOHOHOHOHHOOOOHOHOHOHOHHOOOOHOHOHHOOOOHOHOHOHHOOOOHOHOHHOOOOHOHOHOHHOOHOOHOHOHOHOHHOOHOOHOHOHHOOHOOHOHOHOHHOOOHOHOHOHOH+HOOOHOHOH+HOOOHOHOHOH+OOHCoAsOLeucoantho-cyanidineANSANSFLSFLSFNSFNSCHICHSQuercetin4-Cumaroyl-CoAKaffeoyl-CoAPentahydroxychalkonEriodictyolPentahydroxy-flavanonLuteolinDihydro-myricetinLeucodelphinidinLeucocyanidinANSDFRDelphinidinNaringeninTetrahydroxychalkonTricetinApigeninCHSCHIF3‘,5‘HF3‘HFNSFHTF3‘HMyricetinKämpferolDihydro-quercetinDihydro-kaempferolF3‘,5‘HFLSLeucopelargonidinPelargonidinCyanidinFlavonoleFlavoneFlavanoneChalkoneAuroneOHOCoAsOHOHOCoAsOHOHOCoAsOHOHOHOHOCoAs3*Malonyl-CoAOHOOHOHOHOHOOHOHOHOHOOHOHOHOHOHOOHOHOHOHHOOOOHOHOHOHHOOOOHOHOHOHHOOOOHOHHOOOOHOHHOOOOHOHOHHOOOOHOHOHAnthocyanidineDihydro-flavonoleHOOOOHOHOHOHHOOOOHOHOHOHHOOOOHOHHOOOOHOHHOOOOHOHOHHOOOOHOHOHHOOOOHOHOHOHOHHOOOOHOHOHOHOHHOOOOHOHOHOHOHHOOOOHOHOHOHOHHOOOOHOHOHHOOOOHOHOHHOOOOHOHOHOHHOOOOHOHOHOHHOOOOHOHOHHOOOOHOHOHHOOOOHOHOHOHHOOOOHOHOHOHHOOHOOHOHOHOHOHHOOHOOHOHOHOHOHHOOHOOHOHOHHOOHOOHOHOHHOOHOOHOHOHOHHOOHOOHOHOHOHHOOOHOHOHOHOH+HOOOHOHOHOHOH+HOOOHOHOH+HOOOHOHOH+HOOOHOHOHOH+HOOOHOHOHOH+OOHCoAsOOOHCoAsOLeucoantho-cyanidineANSANSFLSFLSFNSFNSCHICHSMaingroupsofflavonoidsLeucoantho-cyanidineFlavonoidbiosynthesispathwayinappleGoalofthework•OptimizedlevelsofflavonoidsResistanceSignificanceofflavonoidsinresistancetoapplescab(V.inaequalis)andfireblight(E.amylovora)Pigmentation--Fruitquality--Selectionoftransgeniccells(EU:after2008antibioticmarkergenesshouldbeidentifiedandphasedout)ModifyingflavonoidbiosynthesisinappleDownregulationofstructuralgenesbyRNAinterferenceOverexpressionofregulatorygene(maizeLc)I.OverexpressionofmaizeLctranscriptionfactorLcgene•Isolatedfrommaize(Zeamays)•Encodesaproteinsimilartotranscriptionalactivatorsandcontainsthemyc-homologyregion•Responsiblefortissue-specificanthocyanin(flavonoid)production•InducedanthocyaninaccumulationinmaizeI.OverexpressionofMaizeLcTranscriptionFactorTargetsandstrategies•InductionofflavonoidaccumulationinapplewiththehelpoftheLc-gene→resistancetoapplescabandfireblight•Inductionofredcolorationinapple→ThecolorcouldbeappliedasavisualmarkertoselectiontransgeniccellsinappleI.OverexpressionofMaizeLcTranscriptionFactorGenetictransformationInvitro‘HolsteinerCox’TransformationPlasmidwithLcgeneI.OverexpressionofMaizeLcTranscriptionFactorleafexplantsfromnewleavestransformedexplantstransgenicplantMechanismoftransformationThenaturalinfectioncycleofA.tumefaciens(Frandsenetal.,2006)Lc-pBI121constructNOS-proNPTIINOS-proCaMV35SproNOS-prob-GlucuronidaseHindIIIPstISphISmaIXbaIBamHIATGSnaßITGASacIEcoRILBPstIRBSphIPstISphINOS-proNOS-proNPTIINPTIINOS-TerCaMV35SproCaMV35SproNOS-Terb-Glucuronidaseb-GlucuronidaseHindIIIPstISphIHindIIIPstISphISphISmaIXbaIBamHISmaIXbaIBamHIATGSnaßITGASacIEcoRILBPstIRBSphIPstISphICaMV35sProLCNosTerHindIIIEcoRINOS-proNPTIINOS-proCaMV35SproNOS-prob-GlucuronidaseHindIIIPstISphISmaIXbaIBamHIATGSnaßITGASacIEcoRILBPstIRBSphIPstISphINOS-proNOS-proNPTIINPTIINOS-TerCaMV35SproCaMV35SproNOS-Terb-GlucuronidaseLCHindIIIPstISphIHindIIIPstISphISphISmaIXbaIBamHISmaIXbaIBamHIATGSnaßITGASacIEcoRILBPstIRBSphIPstISphII.OverexpressionofMaizeLcTranscriptionFactorPBI121Lc-PBI121Callusregeneration•After6weeksI.OverexpressionofMaizeLcTranscriptionFactorShootsregenerationLCtransgenicplantControlI.OverexpressionofMaizeLcTranscriptionFactorPhenotypeI.OverexpressionofMaizeLcTranscriptionFactorTransgeneintegrationI.OverexpressionofMaizeLcTranscriptionFactorMKLc1Lc2Lc3Lc4Lc6PCRanalysisSouthernblotanalysisGeneexpressionofvectorgenes•nptIILc050100150200250300HCL1L2L3L4L5L6Expression02000400060008000100001200014000160001800020000HCL1L2L3L4L5L6ExpressionI.OverexpressionofMaizeLcTranscriptionFactorPlantmaterial:leavesofinvitroshootsGeneexpressionofflavonoidbiosynthesisgenesThinlayerchromatographyanalysisPKHCLC1LC2LC3LC4LC6CyanidinHydrolicQuercetinNaturalQuercetinNaturalKaempferolCatechinEpicatechinI.OverexpressionofMaizeLcTranscriptionFactorHighperformanceliquidchromatography(HPLC)analysisofflavonoi