烟草侧芽生长相关基因克隆及RNAi载体的构建转化-陈兴江

RNAi550081K3261BLASTGenbankLSMOCLASTLRTLR640bpRNAipHANNIBALNotI4300bppART27TLRpHANNIBAL-TLR-PART27pHANNIBAL-TLR-PART27LBA4404T[e-mail]2012-09-24E-mail:[e-mail]0371-67672650TobaccoPlantAxillaryBudRelatedGeneCloningandRNAiExpressionVectorConstructingandTransformingCHENXingjiang,CAILiuti,andZHENGShaoqingGuizhouTobaccoScienceInstitute,Guiyang550081,ChinaAbstract:Inordertobreedtobaccoplantwithoutaxillarybudgrowingaftertopping,thefragmentDNAsequenceoftobaccoaxillarybudrelatedgene(namedasTLRgene)wasclonedfromflue-curedtobaccocv.K326basingonhomologygenecloningmethod,anditssequencewasanalyzedwithBLASTsearchandGenebankhomologycomparison,itwasfoundthatthatgenehadhighidentitywithLSgeneofLycopersicumesculentum,andhadcertainidentitywithMOCgeneofOryzasativaandLASgeneofArabidopsisthaliana.Thereverseandforwardfragments(640bp)inthereadingframeofTLRgenewereconfiguredintorightandleftsidesofintroninintermediatedRNAivectorpHANNIBAL,respectively.AfterbeingcutbyNotI􀆳afragmentofabout4300bpwasrecoveredandinsertedintoabinaryplasmidpART27,plantexpressionplasmidpHANNIBAL-TLR-PART27withinvertedrepeatedsequencecontainingTLRwassuccessfullyconstructed.ThepHANNIBAL-TLR-PART27wasintroducedintoAgrobacteriumtumefaciensLBA4404,thentransformedcellsintobaccoleaf,butnotransgenicplantwasobtained,itwassuspectedthattheexpressionofTLRgenewasrestrained.Keywords:Axillarybud;Transgenictobacco;RNAi;TLRgene/TobaccoScience&Technology20133308/TobaccoAgronomyls1LS2LSVHIID67LSGAGA3LASGreb4LASRossbergM5LSLi6MOC1MOC1LSLSLASMOC33RNAi11.1NicotiannatabacumK3266DNADNADNeasyPlantMiniKitQiagenpUCM18-TT4DNADNAdNTPDH5αLBA4404pHANNIBALPART271.21.2.1DNADNADNAeasyPlantMiniKit1.2.2LAS1LS4MOC5P15-CSGCKAAYCARGCSATYTTMG-3P25-GCCYTCMGAMGGRTARTGMAG-3DNATLR1.2.3PCRTLRDNAPCR1PCR4000r/min30sPCRPCR955min9430s5250s7260s357210minPCR1.0%EB1.2.4PCRPCRpUCM18-T1.2.5RNAipHANNIBAL-TLR-PART27Varsha7RNAiHannibalpART27pHANNIBALPART27pHANNIBAL1pHANNBIALPCRXhoIEcoRIKpnIClaIHindIlIBarnHIXbaI2INTRON13050bpIhpRNAIhpRNATLRCaMV35STLR-pdk-TLR1TLRcDNAPCR1PCRTab.1PCRamplificationsystemsanditsreagents10×PCRP15μmol/LP25μmol/LDNA25mmoL/LMgCl2dNTP(5μmol/L)Taq(2U/μL)/μL29.55.02.02.01.06.04.00.550.068pHANNIBALXbaIClaIEcoRIXhoI23PCR4000r/min30sPCR955min9430s5250s7240s59430s5530s7240s307210minPCR1.0%EBPCRpUCM18-TpT1pT2RNAi2pHANNIBAL-TLRXhoIEcoRI1%QIAGENEDNA640bppHANNIBALpT1pHANNIBALpT2XbaIClaI1%QIAGENEDNA640bppHANNIBAL-pT1T4DNA3pHANNIBAL-TLR-PART27NotIpHANNIBAL-TLRpPART271%QIAGENEDNA4300bppPART27T4DNAE.coliDH5αpHANNIBAL-TLR-PART271.2.6Clough85mLLB50μg/mLSpe25μg/mLStr25μg/mLRif1∶100LB5h950mg/L600mg/LMS22.1PCR2K326DNAP1P2TLR749bp2.2TLRcDNA3749bpClustalXLASGenbankAY056154.1LSGenbankAJ303345.1MOCGenbankAY242058.1DNAMAN4TLRDNA749bpNCBI-BLASTnGenbank2Tab.2PrimersanditssequencesP1P2P3P4(5-3)ACTTCTAGACGATAACCAACAAGCGATCACTATCGATGATCTGAATCTCTCGTGTCTACTCTCGAGCGATAACCAACAAGCGATCACTGAATTCGATCTGAATCTCTCGTGTCT3PCRTab.3PCRamplificationsystemsanditsreagents10×PCRP1(P3)5μmol/LP2(P4)5μmol/LDNA25mmoL/LMgCl2dNTP(5μmol/L)Taq(2U/μL)/μL24.54.01.01.01.04.04.00.540.069RNAi640bp3TLRDNAFig.3SequenceofTLRfragmentDNAsequence4TLRDNALASLSMOCFig.4TLFgenesequencecomparionwithLAS,LSandMOC83NCBI-BLASTxGenebankTLRGRAS2.3pHANNIBAL-TLR5pHANIBALXhoIEcoRIXbaIClaI2.4pHANNIBAL-TLR-PART276pHANNIBAL-TLRNotIpHANNIBAL-TLR-PART274300bppHANNIBAL-TLR-PART2770HannibalpART2772.5RNAi25mg/L50mg/L1.Da20002.PCR3.XhIEcoRI4.5.XbaIClaI6.5pHANIBALFig.5ResultsofreverseandforwardfragmentsofTLRgenefragmentdegestedbyXhIEcoI,XbaIandClaIrespectively1.Da150002.pHANNIBAL-TLR3.NotI4.pHANNIBAL-TLR-PART275.NotI6pHANNIBAL-TLR-PART27Fig.6ResultsofpHANNIBAL-TLR-PART27vecrorfragmentdegestedbyNotI3TLRNCBI-BLASTGenebankGRASDNARNAi1001200bp10Elbashir11100DNAG-C40%70%TLRG-C59.3%640bpTLR100LSLASMOC13GRAS3GRASA12TLRLSLASMOC13TLRRNAiTLR71201134.14.J.2005142-44.15.J.201143189-94.16.CdD.2010.17.4J.2010181158-163.18.J.2010183654-658.19.J.2010383016888-16890.20.J.2010393453-458.21.CdCdJ.2010294447-451.22.J.2010463346-350.23.J.2012212314-320.24.J.201181411-1421.25SungELMahtabAAdelAetal.EffectsofbiocharonsoilqualityandheavymetalavailabilityinamilitaryshootingrangesoilinKoreaJ.SoilsandFertilizersSciencesinSouthKorea201144167-77.26.J.2008272795-800.27.J.2006432233-239.28ParkJChoppalaG.BiocharreducesthebioavailabilityandphytotoxicityofheavymetalsJ.PlantandSoil20113481439-451.29.J.2009151121-126.TLRRNAi1WilliamsW.TheeffectofeslectiononthemanifoldexpressionofthesuppressedlateralgeneinthetomatoJ.Heredity196014285-296.2MalayerJCGuardAT.AcomparativedevelopmentalstudyofthemutantsideshootlessandnormaltomatoplantsJ.AmerJBot196451140-143.3SchumacherKSchmittTRossbergMetal.TheLateralsuppressorLSgeneoftomatoencodesanewmemberoftheVHIIDproteinfamilyJ.ProcNatlAcadSci199996290-295.4GrebTClarenzOSchaferEetal.MolecularanalysisoftheLATERALSUPPRESSORgeneinArabidopsisrevealsaconservedcontrolmechanismforaxillarymeristemformationJ.GenesDev20031791175-87.5RossbergMTheresKAcarkanAetal.Comparativesequenceanalysisrevealsextensivemicrocolinearityinth