马铃薯腺苷二磷酸的葡萄糖焦磷酸化酶小亚基基因sagp的克隆及其在毕赤酵母中的表达.doc

马铃薯腺苷二磷酸葡萄糖焦磷酸化酶小亚基基因（sAGP）的克隆及其在毕赤酵母中的表达 农业生物技术JournalofAgriculturalBiotechnology2005,13—3):二Z 马铃薯腺苷二磷酸葡萄糖焦磷酸化酶小亚基 基因(sAGP)的克隆及其在毕赤酵母中的表达;lc ? 研究论文? 宋波涛柳俊谢从华成善汉 (华中农业大学园艺林学学院,国家蔬菜改良中心华中分中心,园艺植物生物学教育部重点实验室, 湖北省马铃薯工程技术研究中心,武汉430070) 摘要:用RT.PCR结合RACE的方法,在腺苷二磷酸葡萄糖焦磷酸化酶小亚基基因(sAGP)的保守位点设计l对引物,从低 温处理的马铃薯(SolunumtaberosusL.)块茎cDNA中,扩增出l条约1.9kb的特异性条带,并将其克隆到pBluescriptSK载 体.DNA序列测定结果表明,目标片段为sAGP,全长为l854bp,包含一个l563bp的开放阅读框架,编码521个氨基酸.5’端 有63bp的非翻译区,3’端包含219bp的非编码序列,3’端poly(A)尾端上游l2和l9bp处有加尾信号AATAAA.该基因已在 GenBank注册,登记号为AYI86620.将该基因PCR突变后,克隆到酵母表达载体pMETet.B中,构成表达载体pMETetA4,将其 线性化,用电穿孑L法导入Pichia.methabolicapMADI6,得到重组表达酵母,经甲醇诱导表达后,SDS.PAGE电泳显示,在诱导表 达重组酵母中检测到50kD左右的特异蛋白带.裂解酵母菌体提取蛋白质,进行酶活力活性测定,结果表明诱导表达重组酵母 中AGPase活性明显上升,证明诱导表达成功. 关键词:马铃薯sAGP克隆;酵母表达;酶活性 CloningoftheSmallSubunitcDNAofADPGlucosePyrophosphorylase fromPotatoandItsExpressioninYeastPichia-methabolica SONGBo?TaoLIUJunXIECong—HuaCHENGShan—Han (HuazhongBranchCentralofNationalCenterforVegetableImprovementCentral KeyLaboratoryofHorticulawMPlantBiology,MinistryofEducation;PotatoEngineeringand TechnologyResearchCenterofHubeiProvince,HuazhongAgriculturalUniversity,Wuhan430070,China) b咖ct:ThecompletesAGPcDNAfrompotato(SolariumtuberosusL.)tuberofcloneJHstoredinlowtemperaturewasampli fledusingRT?PCRcombinedwithrapidamplificationofcDNAend(RACE)strategyaccordingtothefragmentofsAGPconservative site,whichyieldeda1.9kbspecificbandthatwasinstertedintothevectorpBluescriptSKII.Thesequenceanalysisshowedthatthe clonedcDNAhadalengthof1854bp,encodingaproteinof521aminoacidsandhada65bp5’untranslatedsequenceanda219bp 3’non?codingregionincludingtwoputativepolyadenylationsignalsAATAAat12and19bpupstreamofthepoly(A)(GenBankAc. cessionnumber:AYl86620).TheclonewassubclonedintotheexpressionvectorpMETct.B.Therecombinantplasmidwastrans. formedintothePichia’methabolicapMAD16.TheresultsofSDS?PAGEanalysisindicatedthatsAGPexpressedinthereceptorand theAGPaseactivitiesofinducedrecombinantyeastwereincreasedsignificantly. rw岫:potatosAGPcloning;yeastexpression;AGP