轉基因生物405918384課件

2024/3/14轉基因生物405918384

11.3Transgenicplants11.3.1WhytransgenicplantsPossibletargetsforcropplantimprovement

TargetBenefit'sDiseaseHerbicideresistanceInsect,Virus

ColdDroughttoleranceSalt

Reductionofphotorespiration

Nitrogenfixation

NutritionalvalueStorageproperties

Consumerappeal

Improveproductivityofcropsandreducetheirlossduetobiologicalagents

Permitgrowthofcropsinareasthatarephysicallyunsuitableatpresent

Increaseefficiencyofenergyconversion

ConferabilitytofixatmosphericnitrogentoawiderrangeofspeciesImprovenutritionalvalueofstorageproteinsbyproteinengineeringExtendshelf-lifeoffruitsandvegetables

Makefruitsandvegetablesmoreappealingwithrespecttocolour,shade,size外植體:植物轉基因的受體外植體的選擇:優(yōu)先葉片、子葉、胚軸等年齡和最佳感受態(tài)期轉化體易于組織培養(yǎng)，易再生易轉化分生組織感受態(tài)細胞所在的部位及數(shù)量用于轉化的植物細胞需具備：高效穩(wěn)定的再生能力與分化和生理狀態(tài)有關,一般要高于80%較高的遺傳穩(wěn)定性穩(wěn)定的外植體來源對選擇性抗生素敏感，便于篩選對農(nóng)桿菌敏感,能有效接受外源基因用于轉化的植物細胞包括：愈傷組織再生系統(tǒng)經(jīng)脫分化、誘導愈傷組織、分化培養(yǎng)獲得再生植株轉化效率高,但穩(wěn)定性差直接分化再生系統(tǒng)不經(jīng)脫分化階段轉化頻率低原生質(zhì)體再生系統(tǒng)周期長,再生頻率低胚狀體再生系統(tǒng)體細胞或單倍體細胞培養(yǎng)誘導而成轉化效率高,可生產(chǎn)人工種子生殖細胞受體系統(tǒng)如花粉、卵細胞轉化效率高11.3.2Methodsforintroducing

clonedDNAintoplantcells1)

Physicalmethods：MicroinjectionBiolisticDNAdeliveryAbovebeusedforbothtransientandstabletransformation2)

Biologicalmethod

APlantvirusescandidatesUntilrecentlywithoutgreatsuccess,beingthefactthatthevastmajorityofplantviruseshavegenomesnotofDNAbutofRNA,ratherdifficultinmanipulationofRNAOnlytwoDNAvirusisknownandneitherisideallysuitedforgenecloningCaulimoviruses,Cauliflowermosaicvirus,CaMV,花椰菜花斑病毒Geminiviruses，GMV,番茄金花葉病毒

RNAviruse(TMV,Tobaccomosaicvirus,andPVX,PotatovirusX)Thevirusesofplantsneverintegrateintothegenomeandarenottransmittedthroughseeds,sostabletransformationcannotbeachieved,however,plantvirusesoftencausesystemicinfectionsB

ThemostwidelyusedarebasedonTiplasmidsfromAgrobacteriumtumefaciens,resultinginthestabletransformationoftheinfectedcell,andthetransferredDNAbehavesasanewgeneticlocusC

Riplasmid植物轉基因的其它分類農(nóng)桿菌介導的基因轉移以原生質(zhì)體或細胞為受體的直接基因轉移種質(zhì)系統(tǒng)的基因轉移,如子房、花粉管等DNA涂于授粉柱頭….經(jīng)花粉管通道,外源DNA經(jīng)珠心到胚囊,達到遺傳轉化stop11.3.3Tiplasmid1)

OverviewofTiplasmidplasmidcontainedinthesoilbacteriumAgrobacteriumtumefaciens,whichinvadesplantsthroughwoundsandinducescrowngalls(tumors)140-235kbTistandsfortumorinducing環(huán)狀dsDNAcrowngalls細胞可分為:章魚堿型、胭脂堿型、農(nóng)桿堿型、農(nóng)桿菌素型、琥珀堿型Inadditiontothegenefortumorformation,carryinggenesforvirulencefunctionsandthesynthesisandutilizationsuchtheunusualaasuchas(opines,octopine,章魚堿andnopaline,胭脂堿)etcTi可作為隨機插入元件,篩選和克隆基因T-DNA:theregionofTiplasmidsresponsiblefortumorformation約25kbIntheplant,theTiplasmidDNAintegratesintooneoftheplantchromosomesbasedonT-DNA主要兩大功能:決定腫瘤的形成和形態(tài)控制crowngall的合成TransferoftheDNAsegmentfromtheTiplasmidtoaplantchromosomerequirestwo25-bpsequencesthatflanktheT-DNA,aswellasseveralgeneslocatedintheTiplasmidT-DNA構建Ti質(zhì)?？寺≥d體的途徑取代型Ti質(zhì)粒克隆載體：用大腸桿菌質(zhì)?？寺≥d體取代野生型Ti質(zhì)粒的全部或部分T－DNA序列，構建而成;通過pBR322與Ti質(zhì)?？寺≥d體之間的同源序列重組，將外源基因整合到Ti質(zhì)?？寺≥d體上.中間質(zhì)?？寺≥d體：T-DNA的部分序列插入到大腸桿菌質(zhì)粒克隆載體，通過輔助質(zhì)粒才能進入農(nóng)桿菌－－植物細胞因T-DNA序列不同：分為：CointegrationThebinaryvectorsystem

2)TwoapproachesusingTi-based

plasmidsA.Cointegration通過T－DNA同源序列的共整合，將外源基因插入到Ti質(zhì)粒上B.ThebinaryvectorsystemBasedontheobservationthattheT-DNAdoesnotneedtobephysicallyattachedtotherestoftheTi-plasmidUsingseparateplasmidstosupplythedisarmedT-DNA(mini-plasmids)andthevirulencefunctionsThemini-Tiplasmid(~20kb)istransferredtoastrainofA.tumefaciens(whichcontainsacompatibleplasmid(~170kb)withthevirgenesforvirfunctions)byatriparentalcrossGenesclonedintomini-TiplasmidsareincorporatedintotheplantcellgenomebytranscomplementationTheT-DNAplasmidissmallenoughtohaveauniquerestrictionsiteandtobemanipulatedusingstandardtechniquesTripartiteortriparentalcrossToovercometheproblemsthattheTiplasmidsaretoolargetobedirectedusedasvectors,并且轉化成的腫瘤細胞不能分化再生為植株TherecombinantispresentinoneE.colistrainAconjugation-proficientplasmidinanotherATiplasmidderivative(含有virgene等)ispresentinA.tumefaciensWhenthethreestrainsaremixed,theconjugation-proficient“helper”plasmidtransferstothestraincarryingtherecombinantplasmid,whichisthenmobilizedandtransferstotheAgrobacterium,recombinantthenpermitsintegrationoftheclonedDNAintotheTiplasmid

葉盤法(leafdiscs)，不宜馬鈴薯植株接種共轉化法，人為創(chuàng)傷或針頭注入植物愈傷組織共培養(yǎng)轉化法植物懸浮細胞共培養(yǎng)轉化法原生質(zhì)體共培養(yǎng)轉化法最大優(yōu)點是獲得的轉化植株來自于同一轉化細胞，成功率低于葉盤法11.3.4Ti的外植體轉化方法

1)

Infectionofplanttissuecanbecarriedoutoftenbyusingleafdiscs,fromwhichplantscanberegeneratedeasily2)

TheonedisadvantageoftheTisystemisthatitdoesnotnormallyinfectmonocotyledonous(monocots)plantssuchascereals(wheat,barley,rice,andmaize)andgrasses3)AsA.tumefaciensandA.rhizogenesinfectonlydicotyledonous(dicots)plants,monocotsareoutsideofthenormalhostrange,othermethodscanbeusedtodeliverrecombinantDNAtothecellsofmonocots(e.g.plantembryo)LeafdiscsMethodofleafdiscs

11.3.5RiplasmidFromAgrobacterium

rhizogenesSimilartoTiplasmid,butresultsnotinacrowngallbutinhairyrootdisease

Thepossibilityofgrowingtransformedrootsathighdensityinliquidculturehasbeenexploredasapotentialmeansofobtaininglargeamountsofproteinfromgenesclonedinplants11.3.6Cloninggenesinplantsbydirect

genetransferComparedwiththeAgrobacteriummeans,directgenetransfersuchasbiolisticstakestheprocessonestepfurtheranddispenseswiththeTiplasmidaltogetherSupercoiledplasmidDNAisusedForbiolistics,plantembryoisfrequentlyused,forothers,protoplastsorsinglecellsmaybeusedProtoplastscanalsobefusedwithDNA-containingliposomesorintactcellscanbevigorouslyshakenwithDNA-coatedsilicaneedles,whichpenetratethecellwallandtransfertheDNAintotheinterior1983年，通過農(nóng)桿菌獲得第一例轉基因植物---轉基因煙草

11.3.7Puttingthetechnologytowork

Transgenicplanttechnologyhasbeenusedforanumberofyears,withvaryingdegreesofsuccess.OneofthemajorproblemsisthepublicacceptanceoftransgenicplantsSomepharmaceuticalrecombinanthumanproteinsexpressedinplantsystem日本農(nóng)水省生物資源研究所等單位將人乳腺中產(chǎn)生乳鐵蛋白的基因?qū)敕哑贩N“秋玉”中開發(fā)出一種基因重組番茄。該番茄能生產(chǎn)母乳中所含的具有提高免疫機能和防止感染的作用、具有增加鐵質(zhì)功效的多功能蛋白質(zhì)－乳鐵蛋白抗蟲基因抗病基因其他基因Bt毒蛋白基因病毒外殼蛋白基因調(diào)節(jié)細胞滲透壓的基因抗鹽堿、干旱蛋白酶抑制基因病毒的復制酶基因

抗凍蛋白基因淀粉酶抑制基因幾丁質(zhì)酶基因

抗除草劑基因植物凝集素基因抗毒素合成基因富含lys的蛋白質(zhì)編碼基因，延熟基因，花青素基因11.4Transgenicanimals11.4.1ConceptofTransgenicanimals外源基因?qū)雱游锏氖芫鸦蚰遗呒毎?，并在細胞基因組中穩(wěn)定整合，再將合格的重組受精卵或囊胚細胞篩選出來，采用借腹懷孕法寄養(yǎng)在雌性動物（fostermother）的子宮內(nèi)，使之發(fā)育成為具表達目的基因的胚胎動物，并能傳給下一代。11.4.2Someofthemethodsforthe

introductionofgenesintoembryosDirecttransfectionorretroviralinfectionofembryonicstemcellsfollowedbyintroductionofthesecellsintoanembryoattheblastocyst(胚泡)stageofdevelopment；Retroviralinfectionofearlyembryos；DirectmicroinjectionofDNAintooocytes,zygotesorearlyembryocells；Sperm-mediatedtransfer；Transferintounfertilisedova；Physicaltechniquessuchasbiolisticsorelectrofusion；Nucleartransfer(usedinorganismalcloning)。Methodsforproducingtransgenicmammals11.4.3Pelementsascloning

vectorforDrosophilaInDrosophila,noplasmidsareknownandcloninginDrosophilamakesuseofatransposoncalledthePelement；Aswellasmovingfromonesitetoanotherwithinasinglechromosome，Pelementscanalsojumpbetweenchromosomes,orbetweenaplasmidcarryingaPelementandoneofthefly’schromosomes。

11.4.4Cloninginmammals1)

WhynotmutagenesisapproachThemutagenesisapproachisnotsuitableforhigherorganismsasthediploidnatureofeukaryoticgenomeanddifficultyintheisolationofanexperimentallyinducedchangeinbothcopiesofagivengeneToovercomethedifficulty

targetedmutagenesisviahomologousrecombination2)

ReasonsforgenecloninginmammalsAtpresent,genecloninginmammalsiscarriedoutforoneofthreereasons:GeneknockoutindeterminingthefunctionofanunidentifiedgeneProductionofrecombinantproteinorpharmingGenetherapySomerecombinantproteinsproduced

inthesecretionsofanimalbioreactors

Somerecombinantproteinsthatareusedtherapeutically

3)Cloningvectorsformammals

SV40

Adenoviruses

Papillomaviruses

Retroviruses4)

GenecloningwithoutavectorMicroinjectionisthemosteffectivewayoftransferringnewgenesintonucleiofmammaliancellsandresultingintheDNAbeinginsertedintochromosomes；TheabilitytointroduceDNAintothegermlineofmiceisoneofthegreatestachievementsofthe20thcenturyandhaspavedthewayforthetransformationofothermammals；

EarlysuccesswasachievedbyinjectingDNAintooneofthepronucleiofafertilisedegg(受精卵)justpriortothefusionofthepronuclei(whichproducesdiploidzygote)tocreatethe“supermouse‘in1982,oneofthemilestonesofgeneticengineering；Ingeneratingatransgenicanimal,itisdesirablethatallthecellsintheorganismreceivethetransgene.Presenceofthetransgeneinthecellsoftheorganismwillenablethegenetobepassedontosucceedinggenerations,andthisisessentialiftheorganismistobeusefulinthelongterm。

(a)Fertilisedeggswereremovedfromafemale；(b)theDNAcarryingtheratgrowthhormonegenewasinjectthemalepronucleus；(c)Theeggswerethenimplantedintoafostermother；(d)SomeofthepupsexpressedtheMGHconstruct(MGH+),andwerelargerthanthenormalpups。Productionof'supermouse'Consequencesofsupermouse證實了“中心法則”在哺乳動物體內(nèi)仍然適用；物種之間的生殖隔離被打破；找到了一條按人類意志定向改造哺乳動物性狀的有效途徑；有了一套集分子、細胞和活體動物水平于一體的全新綜合研究體系。Othertransgenicanimals在小鼠成功后，人們開始進行轉基因家兔、豬、羊和牛等，技術路線基本相似豬是理想動物：來源方便，基因組與人類相近，成功率高于牛羊，一胎多仔，妊娠時間短轉基因家畜生產(chǎn)藥物促進生長、品種目標載體的構建，均通過同源重組方式定點整合到基因組中置換型插入型采集源自小鼠胚泡內(nèi)層的ES細胞并培養(yǎng)防止分化含有突變拷貝基因的DNA導入專門的ES細胞中大多采用電穿孔法5）Proceduresofgene

knockoutinthemouseES細胞Embryonicstemcell，從胚泡內(nèi)細胞團（innercellmass，ICM）分離得到的多潛能細胞確定細胞中突變拷貝基因已取代正?？截惖幕蛱崛S基因組DNA，DNA雜交篩選，應獲得10個以上的克隆中期染色體分析，放棄染色體異常克隆含有一個突變基因拷貝的細胞注入早期胚胎（胚泡）的ES細胞中獲得的是嵌合體小鼠，部分組織或器官來自工程ES，部分來自本身胚胎細胞含有一個突變基因拷貝的鼠相互配對選擇含有兩個突變拷貝基因的鼠Productionoftransgenicmiceusingembryonicstemcelltechnology

11.4.5Applicationoftransgenic

animaltechnologyIntroductionofforeigngenesintoanimalspecies

Ithasbeencarriedout,butinmanycasesthereareundesirablesideeffects,itisclearthatmuchmoreworkisrequiredbeforegeneticengineeringhasamajorimpactonanimalhusbandryThestudyofdevelopmentItisoneareaoftransgenicresearchthatiscurrentlyyieldingmuchusefulinformationByimplantinggeneintoembryos,featuresofdevelopmentsuchastissue-specificgeneexpressioncanbeinvestigatedOneofthemostusefulmousemodelsystemsforinvestigatingembryologicaldevelopmentTheproblemforthisisthatinsertedgenesmaynotalwaysbeexpressedinexactlythesamewayaswouldbethecaseinnormalembryosMiceasanimalmodelsfordiseasestatesOncomouse:PhilipLederandhiscolleagues,HarvardUniversity,Micewereproducedinwhichthec-my