农杆菌介导的AtMYB118基因转化橡胶树易碎胚性愈伤组织体系优化.pdf

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1、南方农业学报JournalofSouthernAgrieuhure2014，45(7)：1137—1146ISSN2095—1191；CODENNNXAABDOI：10．3969／j：issn．2095—1191．2014．7．1137OptimizationofsystemforAgrobacteriumtumefaciens-—-mediatedAtMYBl18genetictransformationintofriableembryog"IllinHbrasiliensiseDryogenlccalllPvPaorasmensls上1BIZheng—hong1

2、，LIZhe2+，WANGZhi—qiul，DAIXue—mei2，FANGJia—lin2，HUANGHua—Slln2，LINWei—fu2，LARDETLudovic3，MONTOROPascal3，CARRONMarc—Philippe3，LECLERCQJulie3，ZUOJian—rll4，MUJin—ye4，YANGXiao—hui4，LIXian95(1c011egeofAgronoIlly，HainanUniversity，Haikou570228，China；2RubberResearchInstitute，CATAS／KeyLaboratory

3、ofRubberBiologyandGeneticResourcesUtilization，Ministr／ofAgriculture／StateEngineeringandTechnologyResearchCenterforKeyTropicalCrops，Danzhou，Hainan571737，China；3EquipeBURST-UMRAGAP，D6partenmntBIOS，CIRAD，Montpellier34398，France；4InstituteofGeneticsandDevelopmentalBiology，CAS，Beijing100101

4、，China；5EnviromnentandPlantProtectionCollege，HainanUniversity，Haikou570228，China)Abstract：【Objective】AnorthogonaldesignL25(56)wasusedtooptimizeAgrobacterium—mediatedAⅢⅧjj8genegenetictranstorelationsystemoffriableemblw-ogeniccalliinHeveabras西eSiSiSinordertoproviderefereneesforgeneticimp

5、rovenmntofitsclones．【Method】Kanamycinataconcentrationof75．0mg／Lwasusedtoselecttrans—tbmledcalli．Theefteetsofsixthetorsviz．，precuhuretime，Agrobacteriumgrowthphase(OD600)，aeetosyringone(AS)concentration，infeetiontime，co—culturetemperatureandco—culturetimeongenetictranstorelationwereeval—

6、uatedbyGUStransientexpressiondetection．【Resuh】SixfactorswerefoundtosignificantlyafteettranstorelationIre—queneyoflong—tel'Illeuhuralfriableemblw-ogeniccalli．Thehighesttranstorelationefficiencywasachievedby0一daypreeuhure，long—tel'IllculturalfriableembryogeniccalliasrecipientsintectedbyA

7、grobacteriumculturescon'espond—ingtoOD6no=0．7for7nlin，followedbyco—culturefor5daysinaco—culturemediumcontaining200Ixmol／LASat25℃．After4—6months，17GUSpositivecalluslineswereachieved．Polymerasechainreaction(PCR)andre—versetranscription—polymerasechainreactionfRT—PCR)analysisresultsoftr