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应用农杆菌介导法将白叶枯病抗性基因Xa21导入水稻光敏不育系培矮64S.docx

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应用农杆菌介导法将白叶枯病抗性基因Xa21导入水稻光敏不育系培矮64S Introduction Riceisastaplefoodformorethanhalfoftheglobalpopulation.However,riceyieldisseverelyaffectedbyvariousbioticandabioticstresses.Amongthebioticstresses,bacterialblightcausedbyXanthomonasoryzaepv.oryzae(Xoo)isoneofthemostseverediseasesthataffectricecrops.Thediseaseischaracterizedbytheappearanceofwater-soakedlesionsthateventuallyturnpaleyellowandthenbrown,leadingtothedeathoftheplant.Onceinfected,yieldscanbereducedbyupto50%. Tocombatbacterialblight,researchershavebeenworkingonthedevelopmentofresistantricegenotypesthatcanresistinfectionbythepathogen.Oneofthepromisingwaysofinducingresistanceinplantsisthroughtheintroductionofresistancegenes.Inthisstudy,wehaveusedAgrobacterium-mediatedtransformationtointroducetheXa21gene,whichencodesareceptor-likekinasethatprovidesresistancetoriceagainstXoo,intoricecultivarPeiai64S. Materialsandmethods Plantmaterial TheplantmaterialusedinthisstudywasricecultivarPeiai64S.Thecultivarwaschosenbecauseofitshighsensitivitytobacterialblight. Bacterialstrain ThebacterialstrainusedinthisstudywasXanthomonasoryzaepv.oryzae(Xoo)strainZhe173.Thestrainwasisolatedfromdiseasedriceleavesandconfirmedaspathogenictorice. Vectorconstruction TheXa21genewasamplifiedfromricecultivarIR24usingPCRandclonedintothebinaryvectorpCAMBIA1300underthecontrolofthemaizeubiquitinpromoter.Thevectoralsocontainedtheselectablemarkergenehygromycinphosphotransferase(hpt)drivenbythepromoterofthecauliflowermosaicvirus35S. Transformationandregeneration TheAgrobacteriumstrainEHA105carryingthebinaryvectorpCAMBIA1300wasusedfortransformation.RicecalluswasinducedfrommatureseedsofPeiai64SonMSmediumsupplementedwith2mg/L2,4-Dand0.5mg/L6-BA.ThecalluswasinfectedwithAgrobacteriumusingaco-cultivationprotocol.After2daysofco-culture,thecalliwerewashedwithsterilewaterandtransferredtoMSmediumsupplementedwith2mg/L2,4-D,0.5mg/L6-BA,500mg/Lcefotaxime,and50mg/Lhygromycin.TheresistantcalliwerethentransferredtofreshMSmediumcontaining1.0mg/LNAA,0.5mg/Lkinetin,500mg/