第六章 植物基因工程

第十章植物基因工程PlantGeneticEngineeringTransgenicPlantsGeneticallyModifiedOrganism(GMO)GeneticallyEngineeredOrganism(GEO)IntroductionThetransgenicplantwasfirstachievedintheearly1980s.Over100differentplantspecieshadbeentransformedThegeneticallyengineeredcharacteristicsconcludeinsectresistance,herbicidetoleranceandimprovednutritionalvaluePlanttissueshowsahighdegreeofdevelopmentalplasticity.PotentialApplicationsof

PlantGeneticEngineeringAgriculturalimprovementsofacropplantUseasabioreactorforproductionofproteinsandmetabolitesAcceleratetraditionalbreedingCreatenovelcharacteristicsThestudyofbiologicalprocessesGeneticallyTransformedPlants第一節(jié)植物遺傳轉(zhuǎn)化的基礎(chǔ)一、植物組織培養(yǎng)技術(shù)1.愈傷組織培養(yǎng)從植物體上切取的小片組織（外植體，explant）放在含有低濃度植物激素的培養(yǎng)基上培養(yǎng)，會(huì)在切口的表面長(zhǎng)出未分化的細(xì)胞團(tuán)——愈傷組織在含有不同激素的培養(yǎng)基中，愈傷組織可分化成根、芽、花或整個(gè)植株2.細(xì)胞懸浮培養(yǎng)當(dāng)愈傷組織被轉(zhuǎn)入液體培養(yǎng)基振蕩培養(yǎng)時(shí)，細(xì)胞團(tuán)會(huì)分散成為單細(xì)胞、小細(xì)胞團(tuán)、大細(xì)胞團(tuán)的懸浮培養(yǎng)物懸浮培養(yǎng)物可無(wú)限培養(yǎng)，但會(huì)發(fā)生聚集遺傳不穩(wěn)定性導(dǎo)致突變的積累，細(xì)胞異源化3.原生質(zhì)體原生質(zhì)體融合原生質(zhì)體轉(zhuǎn)化4.植株的再生Wholefertileplantscanberegeneratedfromtissueexplants，callus，cellsuspensionsorprotoplastsbyplacingthemonappropriatemedia.

DNAcanbeintroducedintomosttypesofplantmaterial—protoplasts,cellsuspensions,callus,tissueexplants,gametes,seeds,zygotes,embryos,organsandwholeplants.TheabilitytorecoveryfertileplantsfromsuchmaterialisoftenthelimitingstepinplantgeneticengineeringratherthantheDNAtransferprocessitself.二、植物基因轉(zhuǎn)移方法AgrobacteriumtumefaciensGeneGunProtoplasttransformationElectroporationMicroinjectionAgrobacteriumTiplasmidwiththenewgenePlantcellcell’sDNATransgenicplantCelldivisionThenewgene+TransformationAgrobacteriumtumefaciens“GeneGun”TechniqueDNAcoatedgoldenparticlesGenegunCelldivisionAplantcellwiththenewgeneTransgenicplantPlantcellCell’sDNADuracellDNAcontainingthegeneofinterestPlantcellProtoplastElectroporationTechniquePowersupplyDNAinsidetheplantcellTheplantcellwiththenewgenePlantTransformationSystems三、植物轉(zhuǎn)化的標(biāo)記基因系統(tǒng)SelectableMarkersReporterGenesPromoterSelectableMarkers

NeomycinphosphotransferasegeneTheantibiotickanamycinisinactivatedbythegeneforneomycinphosphotransferase.AdditionofkanamycintocultureplateswillcauselossofchloroplastfunctionsKanamycinwillselectforthepresenceoftheneomycinphosphotransferasegeneHygromycinphosphotransferasegeneHygromycinisabroadspectrumplantherbicideHygromycin

phosphotransferaseinactivateshygromycinUseofhygromycinincultureplateswillselectforexpressionofthehygromycin

phosphotransferasegeneBar基因膦絲菌素（Phosphinothricin或glufosinate,商品名basta）是一種廣泛使用的除草劑，可抑制谷氨酰胺合成酶的活性，導(dǎo)致植物細(xì)胞內(nèi)氨的致死性累積

鏈霉菌的bar基因編碼的膦絲菌素乙酰轉(zhuǎn)移酶（PAT)使膦絲菌素乙?；ザ拘訮lantReporterGenes報(bào)告基因是用來(lái)篩選和指示轉(zhuǎn)化的細(xì)胞、組織和轉(zhuǎn)基因植株的有效標(biāo)記Plantreportergenesareusedtodetectandquantifygeneexpressioninleaf,root,andflowersCommonreportergenesincludeBeta-Galactosidase,Beta-Glucuronidase(GUS),

luciferases

(fireflyandbacterial),andGreenFluorescenceProtein(GFP)

PlantPromoterSystemsThecauliflowermosaicvirus35S（CaMV）promoteriscommonlyusedasastrongconstitutivepromoterinplantsPromoterstrengthiseffectedbyenhancers,introns,plantspecies,andthesitewithintheplantgenomewheretheT-DNAisinsertedVicilin豌豆球蛋白andphytohemaglutinin植物凝集素,glutenin麥谷蛋白promotersseedspecificexpressionα-amylasepromoterforexpressioninthealeurone糊粉ofcerealgrainsPatatinpromoterfortuberspecificexpressioninpotatoesandtheRuBisCopromoterforgreentissuespecificityOrgan/tissuespecificpromoters第二節(jié)根癌農(nóng)桿菌介導(dǎo)的遺傳轉(zhuǎn)化一、根癌農(nóng)桿菌與Ti質(zhì)粒1.根癌農(nóng)桿菌(Agrobacteriumtumefaciens)

AgrobacteriumtumefaciensA.tumefaciensisagramnegativesoilbacterium

A.tumefaciensinfectsdicotyledonousplantscausingtheformationofCrowngalltumors(冠癭瘤)二、Ti質(zhì)粒（Tumorinducedplasmid）環(huán)狀ds-DNA,160-250kb，具六個(gè)功能區(qū)致瘤區(qū)：合成植物生長(zhǎng)素和細(xì)胞分裂素冠癭堿合成區(qū)：參與冠癭堿合成冠癭堿分解區(qū)：參與冠癭堿分解Ti質(zhì)粒轉(zhuǎn)移區(qū)(tra)：參與在不同農(nóng)桿菌中的接合轉(zhuǎn)移毒性區(qū)（Vir）：直接參與T-DNA的轉(zhuǎn)移和插入植物染色體DNA復(fù)制區(qū)（Rep）：參與Ti質(zhì)粒DNA復(fù)制T-DNA

Ti質(zhì)粒中僅有一部分進(jìn)入植物細(xì)胞，T-DNAT-DNA包括植物激素冠癭堿合成區(qū)基因及兩側(cè)的25bp的正向重復(fù)序列，長(zhǎng)約23kb右側(cè)25bp重復(fù)序列對(duì)T-DNA的轉(zhuǎn)移至關(guān)重要T-DNA可整合到植物染色體上，整合位點(diǎn)不具特異性1.A.tumefacienscellscontainaTi(Tumorinducing)plasmid.HostcellchromosomesGALLFORMATIONISINDUCEDBYAGROBACTERIUMTUMEFACIENS.MainchromosomeTiplasmidT-DNA2.AsectionofDNAfromtheTiplasmid,calledT-DNA,incorporatesintothechromosomesofcellsinfectedbythebacterium.3.Whentranscribed,Tigenesinducetheaffectedcelltobegingrowinganddividing.TheresultinggallprotectsagrowingnumberofAgrobacteriumcells.Ti質(zhì)粒作為載體的優(yōu)點(diǎn)

宿主范圍廣，能轉(zhuǎn)化所有的雙子葉植物整合到染色體上后成為染色體的正常組分永遠(yuǎn)保留冠癭堿合成酶基因啟動(dòng)子是一個(gè)強(qiáng)啟動(dòng)子TiPlasmidCloningVector二、Ti質(zhì)粒載體DisarmedTivectors（非致瘤載體）Cointegratevectors（共整合載體）Binaryvectors（雙元載體）1.DisarmedTivectors去除T-DNA中的腫瘤基因在T-DNA中插入用于轉(zhuǎn)化植株篩選的遺傳標(biāo)記基因IntermediatevectorsAsmallportionofT-DNAwassubclonedinaconventionalE.coliplasmidvector(i.e.pBR322)foreasymanipulation,producingintermediatevectorsIntermediatevectorsareincapableofreplicationinA.tumefaciensandalsolackconjugationfunctions.Transferofthemwasachievedusingatriparentalmating.TriparentalmatingAnE.colistrainAcarryingahelperplasmidabletomobilizetheintermediatevectorintransTheE.colistrainBcarryingtherecombinantintermediatevectorA.tumefacienscarryingthedisarmedTiplasmid3.BinaryvectorT-DNAdoesnotneedtobephysicallyattachedtotherestoftheTiplasmidUseseparateplasmidstosupplythedisarmedT-DNAandthevirulencefunctions

ThesmallplasmidcanbeusedtoinsertedforeigngeneWhenthetwoplasmidspresenttogetherinthesameA.tumefacienscell,theT-DNAcarriedbypBI121istransferredtotheplantchromosomalDNAbyproteinscodedbygenescarriedbypAL4404三、根癌農(nóng)桿菌轉(zhuǎn)化的一般步驟Leaf-disctransformation（葉盤法）四、轉(zhuǎn)基因植物的鑒定報(bào)告基因的檢測(cè)（GUS,GFP……)PCRSouthernblottingNorthernblottingWesternblotting六、發(fā)根農(nóng)桿菌與Ri質(zhì)粒Agrobacteriumrhizogenescausehairy-rootdiseaseinplantsThisisinducedbyroot-inducing(Ri)plasmidOpine-producingroottissueinducedbyRiplasmidinavarietydicotscanberegeneratedintowholeplants第三節(jié)其它的植物轉(zhuǎn)化方法一、原生質(zhì)體轉(zhuǎn)化Polyethyleneglycol（PEG）聚乙二醇ElectroporationThemajorlimitationofprotoplasttransformationistheabilityoftheprotoplastregeneration二、基因槍（particlebombardment）二、基因槍

（particlebombardment）Goldortungstenparticles(0.4to1.2umindiameter)arecoatedwithprecipitatedDNAThecoatedparticlesareacceleratedto300-600meters/seco