28691.家蠶bmstmn基因的表達分析及其亞細胞定位

1、浙江理工大學碩士學位論文家蠶BmSTMN基因的表達分析及其亞細胞定位姓名：李佩申請學位級別：碩士專業(yè)：生物化學與分子生物學指導教師：解純剛張耀洲20100301浙江理工大學碩士學位論文ExpressionAnalysisandSubcellularLocalizationofBmSTMNGenefromSilkworm(Bombyxmori)AbStractStathminisrelatedtotheproliferation，diff

2、erentiationofcellsIthasbeenproposedtobearelayintegratingdiverseintracellularsignalingpathwaysTodate，mostresearchesofstathminwerefocusedonrathervertebratethaninvertebrateThestathmincDNAfromthesilkwormpupacDNAlibrarywasnam

3、edBmSTMN，whoseORFWEtS876bpandencodedaproteinof291anlinoacidsItsmolecularweightcalculatedbytheprogramDNAstarwas3348kD，anditsp1was8005TheresultofMultiBLASTwasthatithadthehighesthomologywithTriboliumcastaneumandthehomologyw

4、as50％TheentireORFWasobtainedbyPC＆andEcoRI脅，刃IⅡrestrictsiteswereaddedbyPCRTheORFWasdigestedbythetwoenzymesandlinkedintopET28a()toconstructrecombinantplasmidpET28《)BmSTMNwhichwasidentifiedbyPCRdoubledigestionandsequencingT

5、herecombinantplasmidWastransformedintoEcoliRosetta(DE3)Afterinducing謝mPTGtheanalysisofSDS—PAGEshowedthatthefusionproteinwasexpressedsuccessfullyinabout37kDlocationTheBmSTMNWasfused、析也HistagandpurifiedonchelatingcolumnsTh

6、emolecularweightofthisfusionproteinwas371910Da(HisTagWasweightof35600DaandBmSTMNWasweightof3348399Da)，characterizedbymassspectrumafterbeingfurtherpurifiedon10R1FPLCThisresultfurtherprovedthatHisTagBmSTMNWasexpressedinthe

7、rightformByimmunizationofNewZealandrabbitwi也purifiedfusionproteinhightiterpolyclonalantibodieswereprepared(1：25600)，andthespecificreactionresponsedverywelldetectedinWesternblottinganalysisSemiquantitativeandwestemblottin

8、ganalysisrevealedthattheproteinwasthehighestexpressedinsilkworm’Spupaandhead，followedbyovaryandtesis，andWasthelowestinsilkglandTheresultofreal—timePCRrevealedthatBmSTMNmRNAwaswidespreadindifferenttissuesandgrowthstagesof

9、silkwormtooconsistent、析ttltheresultsofsemi—quantitativeandwememblottingFinally，wecarriedoutthesubcellularlocalizationaboutBmSTMNinBmNcell，positivesignaltoBmSTMNwasmostlyfoundaroundthemembraneItlaidagoodfoundationforfUlth