马铃薯AGPase小亚基反义cDNA表达和转化载体的构建-生物化学与分子生物学专业毕业论文.docxVIP

摘 摘 要 腺营-二磷酸葡萄糖焦磷酸化酶(AGPase)是细菌糖原和植物淀粉生物合成的关键调控 酶，它催化一磷酸葡钧糖(G—l-P)与ATP合成ADP一葡萄糖和焦磷酸(G-l—P+ATP— ADPG+PPi)，ADP．葡绚糖作为耱供体是淀粉合成酶催化合成a(1-4)或a(1．6)糖苷链形成的底 物。细曲承J植物碳同化中间产物作为变构冈f调控AGPase活性。马铃薯AGPase是由两个 人弧基(LSul和两个小砸基(SSU)组成的异源四聚体，其中人弧墓主要起调¨_作用，小弧基 士要起佳化作Ⅲ。 为了揭示植物贮藏组织淀粉积累调控机理，近年的研究主要集中在AGPase编码基因 的转录调控、AGPase对底物和变构因子敏感性、AGPase翻译后氧化还原调控和异源AGPase 编码基因重组几个方面。已有研究表明反义豁U在储藏组织中表达可抑制淀粉生物合成。 为了在淀粉生物合成突变体内表达外源基因，同时也为了比较不同淀粉生物合成突变体中 AGPase表达的区别，获得淀粉生物合成突变体是先决条件。为此我们开展了本研究，旨在 构建储藏组织专一和非储藏组织专一的反义SSU植物转化载体。为获得淀粉生物合成突变 体奠定基础。 本研究利用分子生物学和基因工程技术：1)分离获得了SSUcDNA；2)构建了反义 鼯驴阵外表达载体；3)构建了分别由CaMV35S和GBSSff苦动子驱动的反义豁罐物转化载 体。结果显示，转化了反义嚣响大肠杆菌株系BL21(DE3)经IPTG诱导后，反gSSU在T7启 动子驱动下表达，糖原含量测定和SDS．PAGE分析表明糖原含量和AGPase亚基平均密度显著 低于对照。这说明反义船嘲大肠杆菌AGPase和糖原的生物合成存在抑制作用． 关键词：AGPase 反义cDNA 反义SSU 表达载体 转化载体 AbstractADPase—glucose Abstract ADPase—glucose pyrophosphorylase(AGPase)is key regulatory enzyme of bacterial glycogen and plant starch biosynthesis．It catalyzes the formation of ADP-glucose from glucose 1．phosphate(GIc-I—P)and ATP with the concomitant release of pyrophosphate(PPi)．The product AGP．glucose then the glucosyl donor for the formation of“14)glucosl chains by glycogen／starch synthase．Both bacterial and plant AGPase activities allosterically regulated by small effecting molecules produced by carbon assimilatory pmhway．AGP∞e in potato is heterotetramer composed oftwo large(LSU，51kD)and two small(SSU，50kO)subunits．The large and small subunits act mainly in part ofregulation and synthesis，respectively． To reveal mechanism of storage starch accumulation in plant，previous investigations of AGPase mainly focused on transcription regulation of genes encoding AGPa．se，sensitivity of AGP笛e to allosteric effectors and substrate，redox regulation ofposttranslation and recombination of heterogenic genes．Meanwhile，studies showed that expression of antisense SSU in storage tissue of transgenic plant led to inhibition of starch biosynthesis．In order to express betemgenic gene in mutants of starch biosynthesis and to disting