甜味蛋白Brazzein在番茄中的遺傳轉化研究.pdf

1、中國農業(yè)科學院碩士學位論文甜味蛋白Brazzein在番茄中的遺傳轉化研究姓名：郭淑華申請學位級別：碩士專業(yè)：蔬菜育種學指導教師：杜永臣王曉武20050601AbstractSweetproteinBra口cinisoneofthenaturalproteinsweetenarsBrazzdnhaslOWcal嘶eandintrinsicsweetness5002000timesov@rsucroseendwillnotcauseweig

2、htgain、toothdecay、andthesugardiabetesthatpeoplesufferedfrequentlywhentakingsugarWeaimedinthepresentstudyatimprovingtomatosweetnesswithoutincreasingthesugarcontentbyintroducingBrazzemgeneintotomatotlmu#transgenictechnique

3、WeclonedafruitspecificexpressionpromotarESPlandconstructedaveetorwhichexpressesasynthesizedBrazzeingeneintomatofruitTransgenicmatctialsⅥre∞obtain。dintwotomatovarietiesZhongshuNo4endLichun1、Developmentofenefficienttomatot

4、ransformationsystem：Weusedl；afdiscandagrobaeterinmmediatedtransformationsystemintomatoThebinary’vectorpBll21Wii8usedaSabasicexpressionvectortomakeconstructsWeselectedpropermediumanddeterminedlOOmgtlkanamycinebeingtheeffe

5、ctiveselectionantibioticconcentrationintransformingtomatoTomatovarieties，MciwciYmgtao，ZhongshuN04endLiChunw啪successfully’transformedwithdifferentconstructsMaintaining100加舭kanamycineinthewholeprocessoftmnsfommtionrodilces

6、ignificantlytheescapesandincreasetheefficiencytodetectpositivemmsfonnentsInthepresentcase，90％ofalltherootedplantswesedetectedpositive2、CloningandfunctionanalysisofafiuitspecificpromoterE8P1WeclonedfromthetomatovarietyHon

7、gmenao213the1121bppromoterE8P1andmadetheGUSexpressingconstructESPl：：GUSTheconstructworesuccessfullytransformedintotomatoPCRtestingofthegenomicDNAandonestepRTPCRverifiedtheintegrationandtheexpressionoftheGUSgeneGUS仃8nscri

8、ptionwasdetectedonlyinthematurefruitsendexpressionwasatahigherlevelthanthatof35S：：GUStransformedplantsinfruitHistochemicaldyingoftheGUSactivityalsoindicatedthatE8PI：：GUStransgenictomatoexpressedGUSspecificallyinmaturefru

9、itThedataprovedthatE8P1couldsoFveasanefficientpromoterforexpressinggenesintomato丘uit3、SynthesizingofBrazzcingeneanditstransformationintomatoWesynthesizedbyPCRa162bpBrazzeingeneusingitscodingsequenceandclonedthePCRfragmen

10、tintoaTvectorWeconstructedtwoplantexpressionvectorspE8P1BandpBll21BforthefruitspecificendconstitutiveexpressionrespectivelyWeconfirmedbyRTPCRthattransgenictomatoesofthetwocons缸uetsexpressedBrazzeingeneatthetranscriptiona