1、寧夏大學(xué)碩士學(xué)位論文TBSV病毒瞬時表達(dá)載體的構(gòu)建及其表達(dá)初步研究姓名：王楊申請學(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：王盛201103IIAbstractThedevelopmentofplantsasbiofactiesftheproductionofproteinproductswithusesinthepharmaceuticalotherindustrieshasflourishedinrecentyears.Pla

2、ntvirusbasedvectsallowftherapidhighleveltransientexpressionofproteinsinwholeplants.Theeticallyexpressionsystemsbasedonviralvectsholdagreatpromise.Althoughmanydifferentplantviruseshavebeenmodifiedtofunctionasexpressionvec

3、tsitishardtosaywhichviralvectisthemosteffectiveonetheexploitationofvirusesasvectsfexpressingfeigngenesstillhavesomethingwhichshouldbeoptimizedimproved.Inadditiontheaccumulationlevelsofdifferentrecombinantproteinsaretotal

4、lydifferentfadesignatedviralvect.Therefefurtherdevelopmentoftheplantvirusbasedvectscouldstillberequired.Tomatobushystuntvirus(TBSV)isthetypespeciesoftheplantinfectingTombusvirusgenusinthefamilyTombusviridae.Overthepastde

5、cadetherehavebeennumerousimptantadvancesinourunderstingofthemolecularbiologyofTBSVsuchasgeneexpressiongenomereplicationrecombination.ThoseadvancesinTBSVmakeitpossibletobeacidateofplantvirusRNAbasedexpressionvect.Furtherm

6、eTBSValsooffersomeparticularadvantagessuchassmallgenomesizefairlyrestrictednaturalhostrangebroadlyexperimentalhostrangecodinggenesilencingsuppressbyitself.FallthesereasonsTBSVhavebeenconsideredanattractivesystemfexpressi

7、ngfeignproductsinplantsshouldbeinvestigatedftheirutilityasgenevects.ConstructsweregeneratedwithfulllengthTBSVcDNAtotalsixTBSVderivedexpressionvects(WY1WY6)whichbelongtothreedifferentcatalogswereconstructed.Thosevectswere

8、deliveredintoTobaccoplants（Nicotianabenthamiana）bymeansofanagrobacteriummediatedtransienttransfmationassay(agroinfiltration)theaccumulationlevelofGFPinagroinfiltratedtobaccoleaveshasbeenanalyzed.Theresultsshowthattheflue

9、scentsignalofGFPcanbedetectedinagroinfiltratedleaves(agroinfiltratedbyrepliconWY1)by2dayspostinfiltration(DPI)maximalaccumulationachievedby1012DPI.PurifiedGFPproteinsareconsistentwithpreviousreptsinmolecularweightfluesce

10、ncespectrumacteristics.TherefeTBSVbasedexpressionvectscanproductfeignproteinintobaccoplantstably.WealsoobservedthattheinoculatedleavesshowobviouslymosaicsymptomsalongwithGFPaccumulation.itdoesntaffectthehostsomuchinperio

11、doftimewhencomparingwithhealthyone.Sothemosaicsymptomsoninfectedplantmayberegardedasadirectffeigngeneexpression.Meanwhiletheresultsalsoshowthattheproteinp22ofTBSVmayhavecrucialrolesduringtheearlyeventsofTBSVinfection.Tos

12、umupweestablishedaTBSVRNAbasedexpressionsystemfstableexpressionofproteinsinNicotianabenthamianaitisproposedthatthisnewtransientexpressionvectwillbeausefultoolfexpressingrecombinantproteinsinplantsfeitherresearchproductio