一种可视化鉴定尾巨桉阳性转化子的基因编辑体系构建研究

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中图分类号：S792.39 文献标志码：A文章编号：1673-923X（2025）10-0218-07

Construction and transformation of CRISPR/Cas9 vector of ALB3 gene inEucalyptusurophylla×E.grandis

LI Limei¹，ZOU Wu1，LIANG Chuyan1，OUYANGLejun12，KE Ziting1， JIANG Bingwei （1.CollegeofloiclnddEegagdogUesittrocicalcholoaogoada 2.GuangdongLingnanCharacteristicPlantGeneResourcesMingandPrecsionEitingEngineeringTchnologyResearchCenteraoming 525000，Guangdong，a;3.AgriclturechologndopularzationCenterofaoingCityaomingouangdoa）

Abstract：【Objective】Eucalyptus hasawiderangeofusesandacomplexgeneticbackground.Geneticengineringresearch can acceleratethegeneticimprovementofeucalyptusandmeet themarketdemandforexcelenteucalyptusvarieties.Establishingan efcientmetodforidentifingpositivetransformantsisaprerequsiteforcaringoutcintgenetictasfomatioandgeeediting inEucalyptus.ALB3gneisacommonlyusedtargetgeneforstudyinggeneediting.TeRUBYreportersystemisanovel，conenient systemthatalowsthedetectiooftereportergenebytenakedeyeundernaturalight.Itiscommonlyusedtargetgeneforifying theeffciencyofplant geneediting.【Method】Polymerasechainreaction （PCR）wasusedtoamplifythegRNAfragmentof heALB3 geneinEucalyptusurophylla ×E grandis.The linearizedPHEE4olE vector was obtained by BsaI enzyme digestion，and therecombinant plasmidpHEE401E-35S-RUBY-ALB3wasconnectedwiththerecombinantenzyme.Therecombinantplasmidwas transformedinto EscherichiacoliDH5asensitivityanalysis，PCRidentificationandsequencinganalysisofheavyparticles，andtransforationof Eucalyptusurophylla ×E grandiscallususingAgrobacterium tumefaciens transformationmethod toidentifythe expressonofreporter genes ineucalptustisueandtheeditingoftagetgenes.【Result】TepHEE4lE-35-RUBY-AB3expressionvctorascostrcted andtransformed intoAgrobacterium tumefaciens.PCRamplificationand sequencinganalysis wereperformedonthe genomeofEucalyptusgrandiscalusithaedphenotypeafterinfectionbyAgrobacteriumtumefaciens.TheresultsshowedthateRUBeporter genehadbeensuccessfullytransferredintothecellandexpressed，andsomebasesquencesofEucalyptusurophylla ×E grandis ALB3 gene was successully kockedoutbetween two targets.The HEE401E-35S-RUBY-ALB3 expresson vector with RUBYsreening markergeneconstructeduinghomologousrecombinationmetodassuccesfullytroducedintocalluscellandthetargetgenewas succesfullyedited.【Conclusio】Tisstudyprovidesacertainreferencefortheestablishmentofanefcientgeneeditingsystemfor Eucalyptusurophylla ×E grandis.

Keywords：CRISPR/Cas9;Eucalyptusgrandis;ALB3gene;RUBY

桉树是桃金娘科桉属树种，具有对环境适应力强、生长速度快的优点，是维护我国木材安全的重要造林树种，也是一种重要的经济林树种[2，经济、社会以及生态效益显著。（剩余11477字）