转基因生物405918384

Chapter11Transgenicplantsandanimals11.1OverviewoftransgenicorganismsTheproductionoftransgenicorganismsinvolvesalteringthegenomesothatapermanentchangeiseffectedItisdifferentfromsomaticcellgenetherapy,transgenicorganismsistoalterthegermline,itisinheritedfollowingreproduction11.2ClassificationofTransgenicorganismsTransgenicplantsTransgenicanimals11.3Transgenicplants11.3.1WhytransgenicplantsPossibletargetsforcropplantimprovementTargetBenefit'sDiseaseHerbicideresistanceInsect,VirusColdDroughttoleranceSaltReductionofphotorespirationNitrogenfixationNutritionalvalueStoragepropertiesConsumerappealImproveproductivityofcropsandreducetheirlossduetobiologicalagentsPermitgrowthofcropsinareasthatarephysicallyunsuitableatpresentIncreaseefficiencyofenergyconversionConferabilitytofixatmosphericnitrogentoawiderrangeofspeciesImprovenutritionalvalueofstorageproteinsbyproteinengineeringExtendshelf-lifeoffruitsandvegetablesMakefruitsandvegetablesmoreappealingwithrespecttocolour,shade,size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11.3.2MethodsforintroducingclonedDNAintoplantcells1)Physicalmethods：MicroinjectionBiolisticDNAdeliveryAbovebeusedforbothtransientandstabletransformation2)BiologicalmethodAPlantvirusescandidatesUntilrecentlywithoutgreatsuccess,beingthefactthatthevastmajorityofplantviruseshavegenomesnotofDNAbutofRNA,ratherdifficultinmanipulationofRNAOnlytwoDNAvirusisknownandneitherisideallysuitedforgenecloningCaulimoviruses,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,plantvirusesoftencausesystemicinfectionsBThemostwidelyusedarebasedonTiplasmidsfromAgrobacteriumtumefaciens,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stop11.3.3Tiplasmid1)OverviewofTiplasmidplasmidcontainedinthesoilbacteriumAgrobacteriumtumefaciens,whichinvadesplantsthroughwoundsandinducescrowngalls(tumors)140-235kbTistandsfortumorinducing环状dsDNAcrowngalls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)etcTi可作为随机插入元件,筛选和克隆基因T-DNA:theregionofTiplasmidsresponsiblefortumorformation约25kbIntheplant,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,aswellasseveralgeneslocatedintheTiplasmidT-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T－DNA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：CointegrationThebinaryvectorsystem2)TwoapproachesusingTi-basedplasmidsA.Cointegration通过T－DNA同源序列的共整合，将外源基因插入到Ti质粒上B.ThebinaryvectorsystemBasedontheobservationthattheT-DNAdoesnotneedtobephysicallyattachedtotherestoftheTi-plasmidUsingseparateplasmidstosupplythedisarmedT-DNA(mini-plasmids)andthevirulencefunctionsThemini-Tiplasmid(~20kb)istransferredtoastrainofA.tumefaciens(whichcontainsacompatibleplasmid(~170kb)withthevirgenesforvirfunctions)byatriparentalcrossGenesclonedintomini-TiplasmidsareincorporatedintotheplantcellgenomebytranscomplementationTheT-DNAplasmidissmallenoughtohaveauniquerestrictionsiteandtobemanipulatedusingstandardtechniquesTripartiteortriparentalcrossToovercometheproblemsthattheTiplasmidsaretoolargetobedirectedusedasvectors,并且转化成的肿瘤细胞不能分化再生为植株TherecombinantispresentinoneE.colistrainAconjugation-proficientplasmidinanotherATiplasmidderivative(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)LeafdiscsMethodofleafdiscs11.3.5RiplasmidFromAgrobacteriumrhizogenesSimilartoTiplasmi