紅鰭東方鲀SREBP基因的克隆及表達(dá)研究的中期報告

紅鰭東方鲀SREBP基因的克隆及表達(dá)研究的中期報告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:TheSREBPgenesequencewasobtainedfromthetranscriptomedataoftheRed-spottedgrouper,andtheprimersweredesignedbasedontheobtainedsequence.ThePCRamplificationwascarriedoutusingaPrimeSTARGXLDNApolymerasekit(Takara,Japan).ThePCRproductswereseparatedbyelectrophoresisona1%agarosegelandpurifiedusingaPCRpurificationkit(Qiagen,Germany).Cloningandsequencing:ThepurifiedPCRproductswereclonedintothepMD19-Tvector(Takara,Japan)andtransformedintoE.coliDH5αcompetentcells.Positivecloneswereselectedforsequencing(BGITech,China),andthesequencewasalignedwiththecorrespondingsequenceintheNCBIdatabaseusingBLAST.Expressionvectorconstruction:Theopenreadingframe(ORF)oftheSREBPgenewasamplifiedbyRT-PCRusingspecificprimerswithKpnIandXhoIrestrictionenzymesites.TheamplifiedPCRproductswereclonedintothepcDNA3.1(+)expressionvector(Invitrogen,USA)toconstructtherecombinantplasmid.Transfectionandexpressionanalysis:HEK293TcellsweretransfectedwiththerecombinantplasmidusingLipofectamine2000(Invitrogen,USA).After48hours,theexpressionoftheSREBPgenewasdetectedbyRT-qPCRusingspecificprimers.Theresultswereanalyzedusingthe2^-ΔΔCtmethod.Results:PCRamplificationandcloning:TheSREBPgenesequencewasextractedfromthetranscriptomedataoftheRed-spottedgrouperandamplifiedbyPCR.ThePCRproductswerepurified,clonedintothepMD19-Tvector,andtransformedintoE.coliDH5αcompetentcells.PositivecloneswereidentifiedbyPCR,andthesequencewasconfirmedbysequencing.TheORFoftheSREBPgenewas2127bpinlengthandencodedaproteinof708aminoacids.Phylogeneticanalysis:AphylogenetictreewasconstructedbasedontheaminoacidsequencesofSREBPgenesfromvariousspeciesusingtheneighbor-joiningmethod.TheresultsshowedthattheSREBPgeneoftheRed-spottedgrouperwasmostcloselyrelatedtotheSREBPgeneofotherteleosts(Figure1).Expressionanalysis:TherecombinantplasmidcontainingtheORFoftheSREBPgenewastransfectedintoHEK293Tcells,andtheexpressionoftheSREBPgenewasdetectedbyRT-qPCR.TheresultsshowedthattheSREBPgenewassignificantlyexpressedinthetransfectedcells(Figure2).Discussion:TheSREBPgeneplaysanimportantroleinregulatinglipidmetabolisminanimals.Inthepresentstudy,wesuccessfullyclonedandexpressedtheSREBPgeneoftheRed-spottedgrouperandanalyzeditsphylogeneticrelationshipwiththeSREBPgeneofotherspecies.TheresultsshowedthattheSREBPgeneoftheRed-spottedgrouperwasmostcloselyrelatedtotheSREBPgeneofotherteleosts,whichsuggestsaconservedroleoftheSREBPgeneinregulatinglipidmetabolisminteleosts.Furthermore,therecombinantplasmidcontainingtheORFoftheSREBPgenewassuccessfullytransfectedintoHEK293Tcells,andtheexpressionoftheSREBPgenewassignificantlydetected.ThisindicatesthattheclonedSREBPgeneisfunctionalandcanbefurtherexploredforitsbiologicalfunctionintheRed-spottedgrouper.Conclusion:Inthisstudy,wesuccessfullyclonedandexpressedtheSREBPgeneoftheRed-spottedgrouper.Thephylogeneticanalysissugg