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水稻APX基因的克隆及其对烟草的遗传转化研究.docx

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水稻APX基因的克隆及其对烟草的遗传转化研究 Abstract: APX(ascorbateperoxidase)isacrucialenzymeinvolvedintheantioxidantdefensesysteminplants.Inthisstudy,weaimedtoclonetheAPXgenefromrice(Oryzasativa)andinvestigateitspotentialforgenetictransformationintobacco(Nicotianatabacum).TheAPXgenewassuccessfullyisolatedfromricebyPCRamplification,andthefull-lengthcDNAsequencewasdeterminedthroughsequencing.ThericeAPXgenewastheninsertedintothebinaryvectorpBI121forAgrobacterium-mediatedtransformationintotobaccoplants.ThetransgenictobaccolinesoverexpressingthericeAPXgenewereobtainedandcharacterizedforvariousphenotypicandbiochemicalchanges.OurresultsindicatedthattheoverexpressionofthericeAPXgeneintobaccoconferredenhancedtolerancetooxidativestressandimprovedoverallplantgrowth.ThesefindingssuggestthatthericeAPXgenehasthepotentialtobeusedingeneticengineeringapproachestoenhancethestresstoleranceofothercropsaswell. Introduction: APXisakeyenzymeintheascorbate-glutathione(AsA-GSH)cycle,whichplaysavitalroleinthescavengingofreactiveoxygenspecies(ROS)inplants.AsROScancausecellulardamageleadingtovariousstress-inducedinjuries,includingoxidativestress,theAPXgeneisessentialformaintainingtheredoxhomeostasisandoverallhealthofplants(Khanetal.,2020).TheAPXgenehasbeenextensivelystudiedinseveralplantspecies,includingrice,anditscloningandgenetictransformationhavebeenreportedtoprovidevaluableinsightsintoplantstressresponsesandpotentialbiotechnologicalapplications(Yuanetal.,2019;Mittleretal.,2004). MaterialsandMethods: 1.CloningofAPXgenefromrice: ThetotalRNAwasextractedfromriceleavesusingacommercialkit,andcDNAsynthesiswasperformedusingreversetranscriptionPCR(RT-PCR).PrimersspecifictotheAPXgeneweredesignedbasedonthericegenomesequenceobtainedfrompublicdatabases.PCRamplificationwasconducted,andtheamplifiedfragmentwasclonedintoaplasmidvectorforsequencing. 2.Genetictransformationoftobacco: Thefull-lengthAPXgenewasexcisedfromtheplasmidvectorandtransferredintothebinaryvectorpBI121.TherecombinantvectorwasintroducedintoAgrobacteriumtume