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1、BreakthroughTechnologiesThepCLEANDualBinaryVectorSystemforAgrobacterium-MediatedPlantTransformation1[W]VeraThole,BarbaraWorland,JohnW.Snape,andPhilippeVain*DepartmentofCropGenetics,JohnInnesCentre,NorwichResearchPark,NorwichNR47UH,UnitedKingdomThedevelopment
2、ofnoveltransformationvectorsisessentialtotheimprovementofplanttransformationtechnologies.Here,wereporttheconstructionandtestingofanewmultifunctionaldualbinaryvectorsystem,pCLEAN,forAgrobacterium-mediatedplanttransformation.ThepCLEANvectorsarebasedonthewidely
3、usedpGreen/pSoupsystemandthepCLEAN-G/pCLEAN-SplasmidsarefullycompatiblewiththeexistingpGreen/pSoupvectors.AsingleAgrobacteriumcanharbor(1)pCLEAN-GandpSoup,(2)pGreenandpCLEAN-S,or(3)pCLEAN-GandpCLEAN-Svectorcombination.pCLEANvectorshavebeendesignedtoenablethe
4、deliveryofmultipletransgenesfromdistinctT-DNAsand/orvectorbackbonesequenceswhileminimizingtheinsertionofsuperfluousDNAsequencesintotheplantnucleargenomeaswellasfacilitatingtheproductionofmarker-freeplants.pCLEANvectorscontainaminimalT-DNA(102nucleotides)consi
5、stingofdirectborderrepeatssurroundinga52-nucleotide-longmultiplecloningsite,anoptimizedleft-bordersequence,adoubleleft-bordersequence,restrictionsitesoutsidetheborders,andtwoindependentT-DNAs.Inaddition,selectableand/orreportergeneshavebeeninsertedintothevec
6、torbackbonesequencetoalloweitherthecounter-screeningofbackbonetransferoritsexploitationfortheproductionofmarker-freeplants.TheefficiencyofthedifferentpCLEANvectorshasbeenassessedusingtransientandstabletransformationassaysinNicotianabenthamianaand/orOryzasativ
7、a.Planttransformationtechnologiesarefundamentaltotheplantnucleargenome,combinedwithanincreasingstate-of-the-artplantmoleculargeneticsandcropim-demandforpreciseandefficienttransformationtech-provementthroughgeneticengineering(Vain,2006).nologies,hascreatedanew
8、opportunitytodevelopplantOverthepast30years,thedevelopmentofnoveltrans-transformationvectorswithimprovedcharacteristics.formationvectorshasbeenseminaltomanybreak-Sincethe1980s,binaryvectorsforAg