转基因生物405918384.ppt

Chapter11Transgenicplantsandanimals,11.1OverviewoftransgenicorganismsTheproductionoftransgenicorganismsinvolvesalteringthegenomesothatapermanentchangeiseffectedItisdifferentfromsomaticcellgenetherapy,transgenicorganismsistoalterthegermline,itisinheritedfollowingreproduction11.2ClassificationofTransgenicorganismsTransgenicplantsTransgenicanimals,11.3Transgenicplants11.3.1Whytransgenicplants,Possibletargetsforcropplantimprovement,外植体:植物转基因的受体外植体的选择:优先叶片、子叶、胚轴等年龄和最佳感受态期转化体易于组织培养，易再生易转化分生组织感受态细胞所在的部位及数量,用于转化的植物细胞需具备：高效稳定的再生能力与分化和生理状态有关,一般要高于80%较高的遗传稳定性稳定的外植体来源对选择性抗生素敏感，便于筛选对农杆菌敏感,能有效接受外源基因,用于转化的植物细胞包括：,愈伤组织再生系统经脱分化、诱导愈伤组织、分化培养获得再生植株转化效率高,但稳定性差直接分化再生系统不经脱分化阶段转化频率低原生质体再生系统周期长,再生频率低胚状体再生系统体细胞或单倍体细胞培养诱导而成转化效率高,可生产人工种子生殖细胞受体系统如花粉、卵细胞转化效率高,11.3.2MethodsforintroducingclonedDNAintoplantcells,1)Physicalmethods：MicroinjectionBiolisticDNAdeliveryAbovebeusedforbothtransientandstabletransformation,2)Biologicalmethod,APlantvirusescandidatesUntilrecentlywithoutgreatsuccess,beingthefactthatthevastmajorityofplantviruseshavegenomesnotofDNAbutofRNA,ratherdifficultinmanipulationofRNAOnlytwoDNAvirusisknownandneitherisideallysuitedforgenecloningCaulimoviruses,Cauliflowermosaicvirus,CaMV,花椰菜花斑病毒Geminiviruses，GMV,番茄金花叶病毒,RNAviruse(TMV,Tobaccomosaicvirus,andPVX,PotatovirusX)Thevirusesofplantsneverintegrateintothegenomeandarenottransmittedthroughseeds,sostabletransformationcannotbeachieved,however,plantvirusesoftencausesystemicinfections,BThemostwidelyusedarebasedonTiplasmidsfromAgrobacteriumtumefaciens,resultinginthestabletransformationoftheinfectedcell,andthetransferredDNAbehavesasanewgeneticlocusCRiplasmid,植物转基因的其它分类,农杆菌介导的基因转移以原生质体或细胞为受体的直接基因转移种质系统的基因转移,如子房、花粉管等DNA涂于授粉柱头.经花粉管通道,外源DNA经珠心到胚囊,达到遗传转化,stop,11.3.3Tiplasmid,1)OverviewofTiplasmidplasmidcontainedinthesoilbacteriumAgrobacteriumtumefaciens,whichinvadesplantsthroughwoundsandinducescrowngalls(tumors)140-235kbTistandsfortumorinducing环状dsDNAcrowngalls细胞可分为:章鱼碱型、胭脂碱型、农杆碱型、农杆菌素型、琥珀碱型Inadditiontothegenefortumorformation,carryinggenesforvirulencefunctionsandthesynthesisandutilizationsuchtheunusualaasuchas(opines,octopine,章鱼碱andnopaline,胭脂碱)etcTi可作为随机插入元件,筛选和克隆基因,T-DNA:theregionofTiplasmidsresponsiblefortumorformation约25kbIntheplant,theTiplasmidDNAintegratesintooneoftheplantchromosomesbasedonT-DNA主要两大功能:决定肿瘤的形成和形态控制crowngall的合成TransferoftheDNAsegmentfromtheTiplasmidtoaplantchromosomerequirestwo25-bpsequencesthatflanktheT-DNA,aswellasseveralgeneslocatedintheTiplasmid,T-DNA,构建Ti质粒克隆载体的途径,取代型Ti质粒克隆载体：用大肠杆菌质粒克隆载体取代野生型Ti质粒的全部或部分TDNA序列，构建而成;通过pBR322与Ti质粒克隆载体之间的同源序列重组，将外源基因整合到Ti质粒克隆载体上.中间质粒克隆载体：T-DNA的部分序列插入到大肠杆菌质粒克隆载体，通过辅助质粒才能进入农杆菌植物细胞因T-DNA序列不同：分为：CointegrationThebinaryvectorsystem,2)TwoapproachesusingTi-basedplasmids,A.Cointegration通过TDNA同源序列的共整合，将外源基因插入到Ti质粒上,B.Thebinaryvectorsystem,BasedontheobservationthattheT-DNAdoesnotneedtobephysicallyattachedtotherestoftheTi-plasmidUsingseparateplasmidstosupplythedisarmedT-DNA(mini-plasmids)andthevirulencefunctionsThemini-Tiplasmid(20kb)istransferredtoastrainofA.tumefaciens(whichcontainsacompatibleplasmid(170kb)withthevirgenesforvirfunctions)byatriparentalcross,Genesclonedintomini-TiplasmidsareincorporatedintotheplantcellgenomebytranscomplementationTheT-DNAplasmidissmallenoughtohaveauniquerestrictionsiteandtobemanipulatedusingstandardtechniques,Tripartiteortriparentalcross,ToovercometheproblemsthattheTiplasmidsaretoolargetobedirectedusedasvectors,并且转化成的肿瘤细胞不能分化再生为植株TherecombinantispresentinoneE.colistrainAconjugation-proficientplasmidinanotherATiplasmidderivative(含有virgene等)ispresentinA.tumefaciensWhenthethreestrainsaremixed,theconjugation-proficient“helper”plasmidtransferstothestraincarryingtherecombinantplasmid,whichisthenmobilizedandtransferstotheAgrobacterium,recombinantthenpermitsintegrationoftheclonedDNAintotheTiplasmid,叶盘法(leafdiscs)，不宜马铃薯植株接种共转化法，人为创伤或针头注入植物愈伤组织共培养转化法植物悬浮细胞共培养转化法原生质体共培养转化法最大优点是获得的转化植株来自于同一转化细胞，成功率低于叶盘法,11.3.4Ti的外植体转化方法,1)Infectionofplanttissuecanbecarriedoutoftenbyusingleafdiscs,fromwhichplantscanberegeneratedeasily2)TheonedisadvantageoftheTisystemisthatitdoesnotnormallyinfectmonocotyledonous(monocots)plantssuchascereals(wheat,barley,rice,andmaize)andgrasses3)AsA.tumefaciensandA.rhizogenesinfectonlydicotyledonous(dicots)plants,monocotsareoutsideofthenormalhostrange,othermethodscanbeusedtodeliverrecombinantDNAtothecellsofmonocots(e.g.plantembryo),Leafdiscs,Methodofleafdiscs,11.3.5Riplasmid,FromAgrobacteriumrhizogenesSimilartoTiplasmid,butresultsnotinacrowngallbutinhairyrootdiseaseThepossibilityofgrowingtransformedrootsathighdensityinliquidculturehasbeenexploredasapotentialmeansofobtaininglargeamountsofproteinfromgenesclonedinplants,11.3.6Cloninggenesinplantsbydirectgenetransfer,ComparedwiththeAgrobacteriummeans,directgenetransfersuchasbiolisticstakestheprocessonestepfurtheranddispenseswiththeTiplasmidaltogetherSupercoiledplasmidDNAisusedForbiolistics,plantembryoisfrequentlyused,forothers,protoplastsorsinglecellsmaybeusedProtoplastscanalsobefusedwithDNA-containingliposomesorintactcellscanbevigorouslyshakenwithDNA-coatedsilicaneedles,whichpenetratethecellwallandtransfertheDNAintotheinterior,1983年，通过农杆菌获得第一例转基因植物-转基因烟草,11.3.7Puttingthetechnologytowork,Transgenicplanttechnologyhasbeenusedforanumberofyears,withvaryingdegreesofsuccess.Oneofthemajorproblemsisthepublicacceptanceoftransgenicplants,Somepharmaceuticalrecombinanthumanproteinsexpressedinplantsystem,日本农水省生物资源研究所等单位将人乳腺中产生乳铁蛋白的基因导入番茄品种“秋玉”中开发出一种基因重组番茄。该番茄能生产母乳中所含的具有提高免疫机能和防止感染的作用、具有增加铁质功效的多功能蛋白质乳铁蛋白,11.4Transgenicanimals,11.4.1ConceptofTransgenicanimals,外源基因导入动物的受精卵或囊胚细胞中，并在细胞基因组中稳定整合，再将合格的重组受精卵或囊胚细胞筛选出来，采用借腹怀孕法寄养在雌性动物（fostermother）的子宫内，使之发育成为具表达目的基因的胚胎动物，并能传给下一代。,11.4.2Someofthemethodsfortheintroductionofgenesintoembryos,Directtransfectionorretroviralinfectionofembryonicstemcellsfollowedbyintroductionofthesecellsintoanembryoattheblastocyst(胚泡)stageofdevelopment；Retroviralinfectionofearlyembryos；DirectmicroinjectionofDNAintooocytes,zygotesorearlyembryocells；Sperm-mediatedtransfer；Transferintounfertilisedova；Physicaltechniquessuchasbiolisticsorelectrofusion；Nucleartransfer(usedinorganismalcloning)。,Methodsforproducingtransgenicmammals,11.4.3PelementsascloningvectorforDrosophila,InDrosophila,noplasmidsareknownandcloninginDrosophilamakesuseofatransposoncalledthePelement；Aswellasmovingfromonesitetoanotherwithinasinglechromosome，Pelementscanalsojumpbetweenchromosomes,orbetweenaplasmidcarryingaPelementandoneoftheflyschromosomes。,11.4.4Cloninginmammals,1)WhynotmutagenesisapproachThemutagenesisapproachisnotsuitableforhigherorganismsasthediploidnatureofeukaryoticgenomeanddifficultyintheisolationofanexperimentallyinducedchangeinbothcopiesofagivengeneToovercomethedifficultytargetedmutagenesisviahomologousrecombination,2)Reasonsforgenecloninginmammals,Atpresent,genecloninginmammalsiscarriedoutforoneofthreereasons:GeneknockoutindeterminingthefunctionofanunidentifiedgeneProductionofrecombinantproteinorpharmingGenetherapy,Somerecombinantproteinsproducedinthesecretionsofanimalbioreactors,Somerecombinantproteinsthatareusedtherapeutically,3)Cloningvectorsformammals,SV40AdenovirusesPapillomavirusesRetroviruses,4)Genecloningwithoutavector,MicroinjectionisthemosteffectivewayoftransferringnewgenesintonucleiofmammaliancellsandresultingintheDNAbeinginsertedintochromosomes；TheabilitytointroduceDNAintothegermlineofmiceisoneofthegreatestachievementsofthe20thcenturyandhaspavedthewayforthetransformationofothermammals；,EarlysuccesswasachievedbyinjectingDNAintooneofthepronucleiofafertilisedegg(受精卵)justpriortothefusionofthepronuclei(whichproducesdiploidzygote)tocreatethe“supermousein1982,oneofthemilestonesofgeneticengineering；Ingeneratingatransgenicanimal,itisdesirablethatallthecellsintheorganismreceivethetransgene.Presenceofthetransgeneinthecellsoftheorganismwillenablethegenetobepassedontosucceedinggenerations,andthisisessentialiftheorganismistobeusefulinthelongterm。,(a)Fertilisedeggswereremovedfromafemale；(b)theDNAcarryingtheratgrowthhormonegenewasinjectthemalepronucleus；(c)Theeggswerethenimplantedintoafostermother；(d)SomeofthepupsexpressedtheMGHconstruct(MGH+),andwerelargerthanthenormalpups。,Productionofsupermouse,Consequencesofsupermouse,证实了“中心法则”在哺乳动物体内仍然适用；物种之间的生殖隔离被打破；找到了一条按人类意志定向改造哺乳动物性状的有效途径；有了一套集分子、细胞和活体动物水平于一体的全新综合研究体系。,Othertransgenicanimals,在小鼠成功后，人们开始进行转基因家兔、猪、羊和牛等，技术路线基本相似猪是理想动物：来源方便，基因组与人类相近，成功率高于牛羊，一胎多仔，妊娠时间短转基因家畜生产药物促进生长、品种,目标载体的构建，均通过同源重组方式定点整合到基因组中置换型插入型采集源自小鼠胚泡内层的ES细胞并培养防止分化含有突变拷贝基因的DNA导入专门的ES细胞中大多采用电穿孔法,5）Proceduresofgeneknockoutinthemouse,ES细胞,Embryonicstemcell，从胚泡内细胞团（innercellmass，ICM）分离得到的多潜能细胞,确定细胞中突变拷贝基因已取代正常拷贝的基因提取ES基因组DNA，DNA杂交筛选，应获得10个以上的克隆中期染色体分析，放弃染色体异常克隆含有一个突变基因拷贝的细胞注入早期胚胎（胚泡）的ES细胞中获得的是嵌合体小鼠，部分组织或器官来自工程ES，部分来自本身胚胎细胞含有一个突变基因拷贝的鼠相互配对选择含有两个突变拷贝基因的鼠,Productionoftransgenicmiceusingembryonicstemcelltechnology,11.4.5Applicationoftransgenicanimaltechnology,IntroductionofforeigngenesintoanimalspeciesIthasbeencarriedout,butinmanycasesthereareundesirablesideeffects,itisclearthatmuchmoreworkisrequiredbeforegeneticengineeringhasamajorimpactonanimalhusbandryThestudyofdevelopmentItisoneareaoftransgenicresearchthatiscurrentlyyieldingmuchusefulinformationByimplantinggeneintoembryos,featuresofdevelopmentsuchastissue-specificgeneexpressioncanbeinvestigated,OneofthemostusefulmousemodelsystemsforinvestigatingembryologicaldevelopmentTheproblemforthisisthatinsertedgenesmaynotalwaysbeexpressedinexactlythesamewayaswouldbethecaseinnormalembryosMiceasanimalmodelsfordiseasestatesOncomouse:PhilipLederandhiscolleagues,Harvard