水楊酸誘導性啟動子的克隆及其在轉(zhuǎn)基因煙草中的表達.pdf

1、中國農(nóng)業(yè)大學碩士學位論文水楊酸誘導性啟動子的克隆及其在轉(zhuǎn)基因煙草中的表達姓名：任海波申請學位級別：碩士專業(yè)：生物化學與分子生物學指導教師：肖興國2002.6.1CloningandExpressioninTransgenicTobaccoofSalicylicAcidInduciblePromoterAbstractInfectionoftobaccowithtobaccomosaicvirus(TMV)resultsinahypers

2、ensitiveresponse(H]R)andleadstosystemicacquiredresistance(SAR)intheplantsTheinductionofSARiSmediatedbysalicylicacid(SA)andiSaccompaniedbymeinducedexpressionofanumberofgenesincludingpathogenesisrelated(PR)genePR1aOffallto

3、baccoPRgenes，PR—la’SexpressionismosthighlyindueedbyTMVandSAtreatmentTherefore，thepromoterofPR—laiSinduciblebysAInthisstudyapmmotcrfragmentof09kbwasobtainedfromtobaccogenomicDNAbyPolymeraseChainReaction(PCR)withthepdmersd

4、esignedaccordingtothePRlapromotersequencereposedinGenbankTheamplifiedfragmentwasclonedintopGEMTvectorandthensequencedSequenceanalysisindicatedthattheclonedsequencewas900bplongandhad99％ofhomologyto也eknownpromotersequenceo

5、fPRJ口ItStranscriptionstartsite，1：f蛆Aboxandconsensussequence‘‘TGAC”conservedinthePRgeneswereidenticaltothoseofPR—ja。InordertostudytheSAindueiblecapacityoftheclonedpromoteracodingsequenceofG【心(pglucurouidase)genewasfusedto

6、itdownstream，andthischimericgenewasclonedintoapBinl9basedplantexpressionvectorcarryingNPTIIgeneasaselectivemarkenThisvectorWasintroducedintotobaccoceilsbyAgrobacleriumtumefaciensmediatedleafdisktransformationandKanresist

7、antplantswereregeneratedSouthernblottinganalysisoftheKanresistantplantsshowedthattheforeigngenesWereintegratedintothegenomeofthetmnsgenietobaccoThechimericgenePRJ口GUSexpressedstronglyintheleavestreated、vitllSAinalltransg

8、eniclinestested，butdidnotexpressorveryweaklyexpressedintheleavestreatedwithwaterbutwithoutSAinsomeoftransgeniclines111echimericGUSgeneexpressedalSOintheentirestems。butnotintherootsoftransgeniclinesofwhichtheleavesweretre

9、atedwithSA，whilewithoutSAinduction，neitherstemnorrootWasGUStrainedIntheuntransformedcontrolplants，nopartswereGUSpositiveTheresultsdescribedabovedemonstratethatthepromoterclonedinthestudyiSSAinduciblepromoterandthispromot