高良姜DXR基因克隆与表达分析.pdfVIP

高良姜DXR基因克隆与表达分析 张春荣1唐晓敏1杨全1 陈虎彪2程轩轩1吴文雅1陈诗敏1 1.广东药学院中药学院，广东 广州 5100062.香港漫会大学中医药学院，香港 [摘要]目的：克隆高良姜1-脱氧-D-木酮糖5-磷酸还原异构酶(DXR)的全长cDNA，分析其组织表达模式，为 通过基因调控和转基因改良高良姜品质奠定基础。方法:应用简并引物RT-PCR和RACE技术从高良姜根茎中克隆 DXR全长cDNA，运用生物信息学解析其编码的蛋白质结构，实时荧光定量PCR法分析其组织表达模式。结果:克 隆了高良姜DXR全长cDNA序列(AoDXR)，开放读码框长1419bp，编码的蛋白质含472个氨基酸残基、相对分子 质量约51.48kDa。推导的AoDXR氨基酸序列与其他高等植物的DXR具有高度的序列相似性(73％～99％)。AoDXR 在高良姜叶片中表达量最强，而在根茎中表达量较弱。结论:AoDXR在高良姜根茎中的表达水平与1,8-桉油精的积 累不一致，反应了AoDXR催化的终产物的多样性和表达调控的复杂性。 [关键词]高良姜；单萜生物合成；1-脱氧-D-木酮糖5-磷酸还原异构酶 Cloningandexpressionof1-deoxy-D-xylulose-5-phosphatereductoisomerasecDNA fromAlpiniaofficinarum ZHANGChunrong1TANGXiaominYANGQuan1CHENHubiao2CHENGXuanxuan1WUWenya1CHENShimin1 1.SchoolofTraditionalChineseMedicines,GuangdongPharmaceuticalUniversity,Guangzhou510006,China;2.School ofChineseMedicine,HongKongBaptistUniversity,HongKong,China [Abstract]TherhizomeofAlpiniaofficinarumHanceisawidelyusedChineseherbalmedicine.TheessentialoilsinA. officinarumrhizomeismainlycomposedof1,8-cineoleandothermonoterpenes,asthemajorbioactiveingredients.Inplants, monoterpenes aresynthesizedthroughthe methylerythritol phosphate (MEP)pathway in the plastids, and 1-deoxy-D-xylulose5-phosphatereductoisomerase(DXR)isanenzymecatalyzingacommittedstepoftheMEPpathway.In thepresentstudy,thefull-lengthcDNAencodingDXRwasclonedfromtherhizomeofA.officinarum,using homology-basedRT-PCRandrapidamplificationofcDNAends(RACE)techniques.ThenewcDNAwasdesignatedas AoDXR.Thefull-lengthcDNAofAoDXRwas1670bpcontaininga1419bpopenreadingframeencodingapolypeptideof 472aminoacidswithacalculatedmolecularmassof51.48kDaandanisoelectricpointof6.15.Bioinformaticanalyses revealedthatAoDXRshowedextensivehomologywithDXRsfromotherplantspeciesandcontainedaconservedplastids transitpeptide,aPro-richregionandtwohighlyconservedNADPH-bindingmotifsinitsN-terminalregioncharacterizedby allplantDXRs.Thetissueexpressionpat