红鳍东方鲀SREBP基因的克隆及表达研究的中期报告-第一文库 | 海量文档资源下载与分享平台

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红鳍东方鲀SREBP基因的克隆及表达研究的中期报告Abstract:ThepresentstudyaimedtocloneandexpresstheSREBPgeneoftheRed-spottedgrouper(Epinephelusakaara)tobetterunderstanditsbiologicalfunction.TheSREBPgenesequencewasextractedfromthetranscriptomedataofthegroupersandamplifiedusingRT-PCR.Theopenreadingframe(ORF)oftheSREBPgenewascloned,andtherecombinantplasmidwastransfectedintoHEK293Tcellstodetectitsexpression.TheresultsshowedthattheORFoftheSREBPgenewas2127bpinlengthandencodedaproteinof708aminoacids.ThephylogeneticanalysissuggestedthattheclonedSREBPgenewascloselyrelatedtotheSREBPgeneofotherteleosts.Furthermore,therecombinantplasmidwassuccessfullytransfectedintoHEK293Tcells,andtheexpressionoftheSREBPgenewasdetectedbyRT-qPCR,whichshowedthattheSREBPgenewassignificantlyexpressedinthetransfectedcells.TheresultsofthisstudyprovideabasisforfurtherexplorationofthebiologicalfunctionoftheSREBPgeneinRed-spottedgrouper.Keywords:Red-spottedgrouper,SREBPgene,cloning,expressionIntroduction:Sterolregulatoryelement-bindingproteins(SREBPs)areafamilyoftranscriptionfactorsthatplayanimportantroleintheregulationoflipidmetabolisminanimals.SREBPsareactivatedbyadecreaseincellularcholesterollevels,whichtriggerstheactivationoftheproteolyticprocessingoftheprecursorprotein.ThematureSREBPtranslocatestothenucleus,whereitbindstothetargetgenepromoterandactivatesthetranscriptionofgenesinvolvedincholesterolandfattyacidsynthesis.TheRed-spottedgrouperisaneconomicallyvaluablefishspecieswidelydistributedinthewatersoftheIndo-Pacificregion.However,themolecularregulationmechanismofitslipidmetabolismisstillunclear.Therefore,weclonedandexpressedtheSREBPgeneoftheRed-spottedgroupertoprovideabasisforfurtherstudyofitsregulationmechanism.MaterialsandMethods:Samplepreparation:Atotalof10matureRed-spottedgrouperswereobtainedfromacommercialfishfarminQingdao,China.TotalRNAwasextractedfromtheliverusingtheRNAisoPluskit(Takara,Japan)followingthemanufacturer'sinstructions.RT-PCRamplification:TheSREBPgenesequencewasobtainedfromthetranscriptomedata