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1CONSTRUCTIONOFPLANTEXPRESSIONVECTORANDTRANSFORMTIONFORATUMEFCIENSOFBMKANEPGENEABSTRACTA1CONCLUSIONCONSTRUCTEDTHEPLANTEXPRESSIONVECTORHARBORINGBMKANEPA2ANTINEUROEXCITATIONPEPTIDEFROMBUTHUSMARTENSIIKARSCHGENE,OBTAINEDENGINEERINGSTRAINBI121DE35SANEPA2A4EHA105FORGENETICTRANSFORMATIONMETHODSUSINGMOLECULARCLONINGTECHNIQUE,INSERTEDANEPA0GENEADDEDKOZAKSEQUENCETO5ENDANDTHESEQUENCEOFERSTRANDEDPEPTIDETO3ENDINTOVECTORPBIN438,ANDFURTHERCLONEDSEQUENCESFROMPBINANEPA0WHICHINCLUDEDCAMVDE35SPROMOTER,ENHANCERANDANEPA0GENETOPBI121GUSINTDTOGETPBI121DE35SANEPA0THERECOMBINANTPLASMIDWASTRANSFERREDINTOATUMEFCIENSEHA105BYFREEZINGTHAWMETHOD,TRIPARENTALMATINGMETHODANDELECTROTRANSFORMATIONTOOBTAINEDTRANSFORMANTRESULTSSUCCESSFULLYCONSTRUCTEDPLANTBINARYEXPRESSIONVECTORPBINANEPA3ANDPBI121DE35SANEPA3A5OBTAINEDENGINEERINGSTRAINPBI121DE35SANEPA3A6EHA105OBJECTIVETOIMPROVEOURPLANTEXPRESSIONSYSTEMTOINCREASETHEEXPRESSIONLEVELOFTHETARGETPROTEINKEYWORDSA7BMKANEPA8,VECTORCONSTRUCTION,PLANTEXPRESSIONVECTOR,ATUMEFCIENSTRANSFECTIONBMKANEP植物表达载体构建及根癌农杆菌转化条件考察A9A10A11A12A13A14A15A16A17A18A19A20BUTHUSMARTENSIIKARSCHA21A22A23A24A25A26A27A28A29ANTINEUROEXCITATIONPEPTIDEA30ANEPA28A31A32A33A34A35A36A30A37A38A39A40A41A42A43A44A45A46A47A48A46A49A50A51A50A51A52A53A54A55A56A57A58PBIN438A59A60A61A62A63A64A65A66A67A68A69A70A71A72A73A74A75A76A66CAMVDE35SA77A78A79A80A81A66A773A82A73A74A83A84A85A86A87A88SEKDELA89A90A91A92ANEPA93A94A92A95A96A97NOSA98A99A100A101A102A103A104A105A106A107A108A109PBI121DE35SANEPA93A110A111A112A113A114A115A116A117A118A119A120A121A122A117A118A123A124A117A125A126A127A128A129A130A131EHA105A104A132A133A134A135A96A136A137A125A126A138A139A92A140A141A110A142A143A144A145A146A147ANEPA93A148A149A150A151A101A102A152A153PBINANEPA93A97PBI121DE35SANEPA93A104A154A155A105A112A156A157A158A125A126A92A159A160A131PBI121DE35SANEPA93A161EHA105A110A142A162A163A164A165A166A167A168A169A170A171A172A173A174A175A176A177A178A179A180A181A182A183A164A184A185A186A187A188A189A167A168A169A170A190A191A192A193A194A195A174A196A197A198A199A200A201A202A203BMKANEPA204A205A206A207A208A209A205A210A211A212A213A206A207A205A214A215A216A217A218A219A220A221A222A223A224A225A226A227A228A229A230A231A226A227A228A229A232A233A234A235A236A205A237A238A239A240A241A242A243A244A245A246A247A244A248A249A250A211A251A232A252A253A207A247A254A207A255A154A250A211A251A232A0A205A155A1A2A3A4A225A245A246A247A225A248A249A16A51A63A42A160A7A8A243A252A9A10A235A10A11A154A115A12A13A212A14A205A169A230A15A17A18A19A20A21A5A22A23A24A4A160A25A26A226A227A228A229A27A28A29A30A8A2114A315A42A241A143A13A32A21A169A230A33A22A233A234A34A35A232A235A236A205A183A36A219A220A37A210A211A0A240A38A18A47A5A210A211A39A40A169A41A230A43A8A211A205A13A18A167A219BMKANEPA192A210A211A44A45A225A234A46A48A10A42A74A49A205A41A239A8A5A162A50A51A52A155A237A210A211A0A45A53A212A2136A42A113A255A154A54A55A106A56A116A57A58A59A60BMKANEPA617A62A63A64A65A66A126A67A59A60A68A62A69A70A71A116A72A73A59A60A75A76A77A78A79A80A81A82CAMVDE35SA83A84A85A86A87A82A62A116A88A89A90A91A92A93A94A95A96A78A79KOZAKA208A97A83A98A99A100A101A102A103A104SEKDELA210A105A107A1088A62A109A110A1112A112A67A114A72A73A59A60A75A76A152A117A118A89A119A120A121A1221材料与方法11A123A124111A125A127A128A129A130A214A131A132A129DH5A133HB101A134A135A136A137A125A127PRK2013A138A133A135A139ANEPA140A141A142A144A218A145A146A147A148A149PNJUANEPA140A150A151A153A156A157A158A159A161A163A164A165A132A129EHA105A133A166A168A170A171A146A147A148A149PBI121GUSINTDA133PBIN438A221A172A173A174A175A176A168A177A178A179A180A181A182A184A185A186112A233A187A176A168A188A189A190PMD19TVECTORA161A236A191A193A194A195A196SALA239A197BAMHIA197HINDA198A197XBAA239A199T4DNAA240A200A196A201A202A203A240A204A205A206A244TAQDNAPOLYMERASEA197TAQPLUSDNAPOLYMERASEA201A202TIANGENA244DNAA245A207A209A211A212A213A201A215AXYGENA244PCRA216A206A217A219A220A222A223A224A225A226A227A244A228A229A211A212A230A231A232A234A235A23712A238A241121A242A243A246A247A248A249ANEPA250A242A243A251A252A253GENEBANKA254A255A6A0A6A1A2A1A15A23A12A3A4A5A62A7A16A21A242A243A8A9A10ANEPA250FA10A17A18A19A11A11A11A254A20A19A19A11A19A19A254A19A19A254A20A11A11A254A20A11A11A254A20A254A20A254A20A254A254A11A254A11A11A254A254A11A20A254A254A19A11A11A254A20A11A18A2A77BAMHIKOZAKBOXANEPA13RA14A22A24A25A26A27A26A27A28A26A27A25A26A28A28A25A28A25A27A26A28A26A25A28A26A28A28A28A26A28A26A25A27A25A26A28A28A28A28A28A27A26A26A25A26A26A27A26A25A28A27A28A25A28A28A25A26A28A28A24A29A30SALA31A32A33A34A35A36A37SEKDELA38A39A40A43A41A42ANEPA13A75A44A45PNJUANEPA13A66A83A46A47A48A201A49A50A51A30A52A84A53A54A46A14A55A56A57TAQDNAPOLYMERASEBUFERA58LA30DNTPSA59A58A60A22MM04LA30A203A61A63A56A60A64L10PMA65A30A47A48A59A58A56NG2LA30TAQDNAA67A254A68A56A60A58L5U/LA30A69A70A71A72A73A74A76A78A79A80A53A81A185A58A56LA82A52A84A85A124A46A14A86A64A87A88A135A58A56A89A30A22A56A87A90A91A29A56A89A30A92A58A87A93A94A64A56A89A30A58A86A173A95A96A244A97A70A92A58A87A93A94A55A56MINA82A183A98A99A45ANEPA13A75A44A100A101A102A103A104PMD19TA105A53A30A106A107A108A109A110A73A111A112A113A114A115DH5A30A71ANEPA13FA116RA194A117A135A203A61A201A49A73A118PC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A230A246A248PCRA249A250A251FIG4,5MA253DL200MARKERMA253DL200MARKERLANE1A253CONTRO1LANE1A253PCRPRODUCTOFANEPA221LANE2A253PCRPRODUCTOFPNJUANEPA221LANE2A253CONTRO1LANE35A253PCRPRODUCTOFPBINANEPA221CLONESLANE36A253PCRPRODUCTOFPBI121DE35SANEPA221CLONESFIG4IDENTIFICATIONOFPBINANEPA255BYPCRFIG5IDENTIFICATIONOFPBI121DE35SANEPA221BYPCR24A24A0A1A2A3A4A5A6A7A8A9A24A0A1A2A4A5A6A7241A85A10A11A12A13A101A14A15A16A17A18A59A85A10A117A11A87A97A59A36A19A20A21A87A97A90A22A23A25A26A27A28A29A96A30A31A177A32A225A33A34A60A35A152PCRA115A61A165A37A38A179A14A152GA16A17A59A85A10A117A112SA31A36A19A20A2160SA44A39A40A41A124A42242A90A43A45A153A13A39A46A47A48A41A124A49EHA105A47A4850HA31ECOLIHB101A50A55A51A52A16A17PRK2013A210A47A4812HA31ECOLIDH5A50A55A53A54A16A17PBI121DE35SANEPA56A210A47A4810HA42243A94A57A13A58A62A63A94A182220KVA31A95A57A87A9755MSA42A84A63A90A22A64A13A58A62A65A66A33A183A67A68A69A70A71A34A72A73A34PCRA115A61A165A37A31A107FIG6FIG3SEQUENCINGRESULTOFTANEPA56BYM13PRIMER6MA74DL200MARKERLANE1A74CONTRO1LANE2A74PCRPRODUCTOFPNJUANEPA56LANE311A74PCRPRODUCTOFPBI121DE35SANEPA56/EHA105CLONESFIG6IDENTIFICATIONOFPBI121DE35SANEPA56/EHA105BYPCR3讨论A113A75A115A66A33A76A77A78A79A80A81A82PBI121DE35SANEPA56A31A184A83A55A86A88A89CAMVDE35SA59A91A92A93A98A89A59A104A99A65ANEPA56A100A26A59GUSA100A26A59NOSA102A103A89A79A80A105A31A189A22A106A108A109A124A76A110A15A38A111A112A150A114A116A118A119A126A120A121A122A123A125A127A128A129GUSA130A131A132A133A134A135A136A186A137A112A132A133A138A139A140A141A202A187A142A143A144A145A202A146A147A148A149A138A139A151A154A155A156A157A158A120A151A154A128A159A160A161A162A163A164A129A166A167A138A139A151A154A156A168A120A169A151A154A170A167A121A122A123A125A190A141A145A141A171A172A173A132A191A142A174A175A176A178A180A112A137A181A185A188A192A193A194A195A129A196A197A198A199A200A195A196A201A180A203A204A205A206A207A211A208A209A212A181A185A213A214A129A215A216A217A209A212A181A185A195A196A201A180A218A219A220A221A222A211A129A223A224A216A226A129A166A227A196A197A198A199A142A228A229A196A201A230A200A129A135A136A231A232A220A216A217A181A185A233A129A234A235A200A112A137A169A236A120A129A215A237A238A237A239A244A196A197A198A199A200A135A157A158A229A240A241A242
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