水稻oryzasatival碳酸酐酶基因在逆境胁迫下的功能解析-functional analysis of rice oryzativar carbonic anhydrase gene under stress.docxVIP

东北林业大学硕士学位论文蛋白19mg。纯化的得到的CA酶活性受到来自于磺胺类物质(Acetazolamide)和阴离子(N，’、N03’、I‘、Br、CI一)的抑制，同时它不具有显著的酯酶活性，Zn2+能够帮助CA更好的保持活性。所获蛋白的质量与数量，足够用于制备抗体，这对今后在蛋白质水平上研究CA与逆境的应答关系奠定了良好的基础。关键词水稻；碳酸酐酶；环境逆境；转基因；原核表达AbstractAbstractCarbonicanhydrase(CA，EC4．2．1．1)，azinc—containingmetalloenzyme，theenzymehasbeenfoundinmostorganismsincludinganimals，plants，archaebacteria，eubacteria．CAplaysanimportantroleinphotosynthesis．ButsomeresearchersfoundthatCArespondedtoenvironmentalstresses．Forcatalyzingthereversibleinter-conversionofC02andHC03’(C02+H20HHC03’+H十)，wefocusedinCarbonatestress(NaHC03，Na2C03)forplantinthealkalisoil(NaHC03，Na2C03)inthenortheastofChina．Usingmolecularbiologicalmethods，therelationshipbetweenCAandstresswasstudiedinriceplant．Thoseresultswereas■followed：1．TheresultsofcomparativeexperimentationshowedthatthegerminationratesandthegrowthofricewereaffectbytheNaHC03andNaClstresses．RicerootsweremoresensitivetoNaHC03thantheshoot．ComparewithNaClstress，Na+，pHvalue，andHC03。weresuppliedbyNaHC03stress．2．TounderstandtheresponsiverelationshiobetweenriceCAandenvironmentalstress，experimentstudiedtheprofileofriceCAexpressionbyNorthrenblotanalysisusingriceseedlingsunderseveralstresses．TheresultsshowedthatthelevelsofCAtranscriptswereincreasedunderstressesfromNaCI，NaHC03，andPEGandtheintensityofsaltanddroughtstressesinfluencedtheexpressionofriceCA．Moreover,CAactivityalsoincreasedunderNaHC03、Na2C03、NaCIandPEG6000stresses．TheseresultssuggestedCArespondedtoenvironmentalstresses．3．InordertoresearchtherelationbetweenCAgeneandenvironmentalstressesbetter,theORFofriceCAwasconstructedintoplantexpressionalvectorpBI121，andintroducedinto,4rabidopsisthalianausinganAgrobacterium—mediatedsystem．TheresultofnorthemblotprovedthattheCAgenewastransferredintotheArabidopsisgenome．TransgenicArabidopsisoverexpressingthericeCAgeneshowedgreatersalttoleranceatseedlingstagethanwild_typeplantsinthe1／2MSmediumwith5mMNaHC03，50mMNaCI，and100mMNaCI．4．TheCAgeneandGFPweretogetherclonedintoplantexpressionalvectorpBI121，thentheplasimedpBI121-