Ti質(zhì)粒與植物基因組的相互作用

Ti質(zhì)粒與植物基因組的相互作用第1頁/共21頁Figure1.Intheprocessofcausingcrowngalldisease,thebacteriumAgrobacteriumtumefaciensinsertsapartofitsTiplasmid—aregioncalled

T-DNA—intoachromosomeofthehostplant.第2頁/共21頁Figure2.SimplifiedrepresentationofthemajorregionsoftheTiplasmidofA.tumefaciens.TheT-DNA,wheninsertedintothechromosomalDNAofthehostplant,directsthesynthesisofnopaline,whichisthenutilizedbythebacteriumforitsownpurposes.T-DNAalsodirectstheplantcelltodivideinanuncontrolledmanner,producingatumor.第3頁/共21頁StructureofTi-plasmidT-DNA區(qū)毒性區(qū)(Vir-region,vir)質(zhì)粒復(fù)制起點(diǎn)(Originofreplication,ori)質(zhì)粒結(jié)合轉(zhuǎn)移位點(diǎn)

(Transferfunctionsite,tra)冠癭堿分解位點(diǎn)(ocsornos)第4頁/共21頁TwotypesofTi-plasmidOCSplasmid(octopine)pTiAch5,pTiA6NC,pTiB653,pTiAg162NOSplasmid(nopaline)pTiT37,pTiT38第5頁/共21頁土壤農(nóng)桿菌-植物DNA轉(zhuǎn)移體系步驟植物敏感細(xì)胞和土壤農(nóng)桿菌相互作用土壤農(nóng)桿菌的毒性區(qū)基因被激活T-DNA的切割和T復(fù)合物生成T復(fù)合物由土壤農(nóng)桿菌經(jīng)植物細(xì)胞膜進(jìn)入植物細(xì)胞T-DNA整合到植物染色體上第6頁/共21頁Figure1.Intheprocessofcausingcrowngalldisease,thebacteriumAgrobacteriumtumefaciensinsertsapartofitsTiplasmid—aregioncalled

T-DNA—intoachromosomeofthehostplant.第7頁/共21頁InteractionofAgrobacteriumTi-plasmidDNAandplantcells

植物敏感細(xì)胞和土壤農(nóng)桿菌相互作用植物受傷產(chǎn)生大量對土壤農(nóng)桿菌感染敏感的細(xì)胞受傷細(xì)胞產(chǎn)生一些低分子量的酚類物質(zhì)可明顯刺激土壤農(nóng)桿菌Ti質(zhì)粒上毒性區(qū)表達(dá)乙酰丁香酮（AS），α-羥基乙酰丁香酮第8頁/共21頁Ti質(zhì)粒毒性區(qū)基因激活及

T-DNA復(fù)合物生成Insertpicture第9頁/共21頁IntegrationofT-DNAintothechromosomeofhostplant

T-DNA整合到植物染色體上首先在植物靶DNA上出現(xiàn)一個缺刻(nick)隨著DNA解鏈，宿主細(xì)胞的5’—3’外切酶活性使缺刻擴(kuò)大成裂口(gap)T-DNA侵入裂口，末端與靶DNA單鏈上的少數(shù)核苷酸配對形成異源二倍體；T-DNA懸掛在外側(cè)的末端被切割除去，T-DNA與靶DNA末端相連；靶DNA上鏈出現(xiàn)缺刻，以整合后的T-DNA下鏈為模板合成T-DNA的第二條鏈，完成整合過程。第10頁/共21頁ReferencesviaNCBITheinteractionofAgrobacteriumTi-plasmidDNAandplantcells(1980)Transgeneintegrationinaspen:structuresofintegrationsitesandmechanismofT-DNAintegration.(2002)HowdoestheT-DNAofAgrobacteriumtumefaciensfinditswayintotheplantcellnucleus?(1993)T-DNAintegration:amodeofillegitimaterecombinationinplants.(1991)TransferandIntegrationofT-DNAwithoutCellInjuryintheHostPlant.(1997)IntegrationofAgrobacteriumT-DNAintoatobaccochromosome:possibleinvolvementofDNAhomologybetweenT-DNAandplantDNA.(1990)Illegitimaterecombinationinplants:amodelforT-DNAintegration.(1991)DNArearrangementassociatedwiththeintegrationofT-DNAintobacco:anexampleformultipleduplicationsofDNAaroundtheintegrationtarget.(1995)TheDNAsequencesofT-DNAjunctionssuggestthatcomplexT-DNAlociareformedbyarecombinationprocessresemblingT-DNAintegration.(1999)Twodifferentbutrelatedmechanismsareusedinplantsfortherepairofgenomicdouble-strandbreaksbyhomologousrecombination.(1996)第11頁/共21頁P(yáng)roductionoftransgenicplantswithrecombinantTi-plasmidFormationofCointegrateTi-plasmidGenerationofatransgeneticplantviathegrowthofacelltransformedbyT-DNA第12頁/共21頁Figure3.(a)Toproducetransgenicplants,anintermediatevectorofmanageablesizeisusedtoclonethesegmentofinterest.Inthemethodshownhere,theintermediatevectoristhenrecombinedwithanattenuated(“disarmed”)Tiplasmidtogenerateacointegratestructurebearingtheinsertofinterestandaselectableplantkanamycin-resistancemarkerbetweentheT-DNAborders,whichisalltheT-DNAthatisnecessarytopromoteinsertion.(b)ThegenerationofatransgenicplantthroughthegrowthofacelltransformedbyT-DNA.第13頁/共21頁FormationofCointegrateTi-plasmidAttenuatethenaturalTi-plasmid.havetheentireright-handregionofitsT-DNA,includingtumorgenesandnopaline-synthesisgenesdeleted,renderingitincapableoftumorformation.Prepareaintermediatevector.Incorporatetheintermediatevector.第14頁/共21頁Fig3.(a)Toproducetransgenicplants,anintermediatevectorofmanageablesizeisusedtoclonethesegmentofinterest.Inthemethodshownhere,theintermediatevectoristhenrecombinedwithanattenuated(“disarmed”)Tiplasmidtogenerateacointegratestructurebearingtheinsertofinterestandaselectableplantkanamycin-resistancemarkerbetweentheT-DNAborders,whichisalltheT-DNAthatisnecessarytopromoteinsertion.第15頁/共21頁GenerationofatransgeneticplantviathegrowthofacelltransformedbyT-DNABacterialinfection

transformedplantcellCellculturationcallusInducingthedifferentiationofshootsandroots,andtransferringtosoil

transgeneticplant第16頁/共21頁Fig.3(b)ThegenerationofatransgenicplantthroughthegrowthofacelltransformedbyT-DNA.infection第17頁/共21頁Figure4.T-DNAandanyDNAcontainedwithinitareinsertedintoaplantchromosomeinthetransgenicplantandthentransmittedinaMendelianpatternofinheritance.第18頁/共21頁ViaNCBI,fromModernGeneticAnal