Isolation of Nile Tilapia （Oreochromis niloticus） β-actin Promoter and Assay of Its Transcription Activity.pdfVIP

强 ScienceDirect December2O13 V0l_2ONo．4 64—71 JournalofNortheastAgriculturalUniversity(EnglishEdition) Availableonlineatwww．sciencedirect．cam IsolationofNileTilapia(OreochromisnfloOcus)一actinPromoterand AssayofItsTranscriptionActivity WangMao．yuan，_，YangHong，ZhongQuan-fu2，LaiMing。yong2，andFanHai—ping2 FreshwaterFisheriesResearchCenter，ChineseAcademyofFisherySciences，Wuxi214081，Jiangsu，China F~inaInstituteofAqucticProductinFreshwater，Fuzhou350002，Chma Abstract：ThroughPCRamplification，5’flankingregionandpartialopenreadingflame(ORF)ofgeneofNiletilapia(Oreochromis niloticus)wasclonedbyPCRamplification．Sequenceanalysisshowedthatnodifferencewasfoundinknownfunctionalregions． ThisstudywastoconstructandidentifythemammalianexpressionvectorofpEGFP-fl—actinandtodetectwhetheritcouldexpressin HEK 293T eellline．pEGFP ．actinwastransfectedintoHEK 293T cellswiht Lipofectamine2000．Theresultsshowedhtatcorrect constructionofrecombinantpEGFP -actinhasbeenshownbyrestrictionenzymedigestion．TheexpressionofgeneinHEK 293T cellscouldbeobservedundermicrofluoroscope．pEGFP-fl-actincouldrepressEGFPproteininHEK293Tcells．Theresultsshowed thatfl-actingenepromoterpossessedeffectivetranscriptionactivitiesineukaryoticcells．Theworklaidfoundationsforfurtherstudy onthegeneengineeringandauto~nasgenictilapia． Keywords：Oreochromisniloticus， actin，promoter，greenfluorescentprotein，rtansfection CLCnumber：Q959．46 Documentcode：A ArticleID：1006—8104(2013)一04—0064—08 geneticengineeringmainlyderiverfom promoters Introduction ofCyprinuscarpio 一actingene(Liueta1．，1990)， Macrozoarcesamericanusantifreezeproteingene Theisolationandidentificationofpromotersisasigni· (Dueta1．，1992)，Daniorer