重組牛乳鐵蛋白抗菌肽基因在煙草中的表達(dá)及功能研究.pdf

1、中國農(nóng)業(yè)大學(xué)碩士學(xué)位論文重組牛乳鐵蛋白抗菌肽基因在煙草中的表達(dá)及功能研究姓名：樊立申請學(xué)位級別：碩士專業(yè)：生物化學(xué)與分子生物學(xué)指導(dǎo)教師：王國英20030601AbstractLactoferrin，amemberoftransferrinfamily，isamultifunctionalglycoproteinThelactoferrincin(Ifcin)’apeptidederivedfromthebovinelactoferrin

2、，exibitesstronglyincreasedantibacterialabilityInthisstudy，arecombinantlfcingenehasbeensynthesizedandusedfortobbacotransformationMolecularevidencedemonstratedthatlfcingenehadbeentransformedintothegenomeoftransgenictobacco

3、FunctionalanalysisconfirmsthenaturalantibioticactivityandelevatedperformanceoftransgenictobbacoplantstodefensethemselvesfromtheTMVinfectionThepreliminaryresultsa佗越foIlows1CodondesignandgenesynthesisInordertoimprovetheexp

4、ressionofLfcinBinplant，wemodifiedsomecodonsaccordingtothecodonusagebiasofplantSynthesizedsinglestrandDNAwereannealedandstretchedAstartcedonATGwasaddedatthebeginningoftheORFandthestopcodonattheendofitTheproductofPCRwasclo

5、nedintoTEasyvectorandsequenced2FusionexpressionofIfcininEcoliLfcinwasclonedintotheexpressionvectorofpMALc2X，whichisafusionexpressionsystemTheresultsofsequencingshowedthatIfcinhadintegratedintothedownstreamofMBPgeneSDSPAG

6、EelectrophoresisandWesternblottinganalysisconfirmedthelowexpressionlevelofLfcininECO豇3TransferofLfcinintotobaccoandtheanalysisoftransgenicplantsLfcinwasfusedtoasignalpeptidegenederivedfrompotatoproteinaseinhibitorIIandin

7、corporatedintop3301plasmidatNcoIandBstEIIsitesinsteadofGUSgene，andthentransferredintotobaccousingagrobaeterium—mediatedstrategyTransgenicplantswereconfirmedbymolecularanalysisincludingPC凡SouthernblotandRTPCR4Functionalan

8、alysisCrudeproteinsoftransgenictobaccoWasextractedfromtheleavesthenusedforthetestofLfeinantibioticactivitiesInthestudy，EcoliDH5aandStaphylocoddusaureaswereservedastargetmicrobesThegrowthofbothbacterialswerestronglyinhibi

9、tedbytheproteinextractsInaddition，transgenictobaccocouldalleviatemosaicsymptomsintobaccoleavesinoculatedwjthtobaccomosalevirus5CloningofrabbitbreastlactoferringeneByusingofthekitsofRACE，wetrytoclonetherabbitlactoerringen

10、efromthebreastmaterialsWeonlyclonedthemRNAoftransferrinbecauseofthehighhomogeneitybetweenthelactoferrinandtransferrinproteinsKeywords：lactoferringene，lactoferrincin(Lfcin)，genetictransformation，tobacco，antibioticactivity