马铃薯腺苷二磷酸的葡萄糖焦磷酸化酶小亚基基因的克隆及其在.doc

马铃薯腺苷二磷酸葡萄糖焦磷酸化酶小亚基基因()的克隆及其在 Cloning of the Small Subunit cDNA of ADP Glucose Pyrophosphorylase from Potato and Its Expression in Yeast SONG Bo-Tao LIU Jun** XIE Cong-Hua CHENG Shan-Han The complete cDNA from potato L. tuber of clone JH stored in low temperature was ampli- fied using RT-PCR combined with rapid amplification of cDNA end RACE strategy according to the fragment of conservative site, which yieldeda 1.9 kb specific band that was insterted into the vector pBluescript SK域. The sequence analysis showed that the cloned cDNA hada length of1 854 bp, encodinga protein of 521 amino acids and hada 65 bp 5 untranslated sequence anda 219 bp 3 non-coding region including two putative polyadenylation signals AATAA at 12 and 19 bp upstream of the polyA GenBank Ac- cession number: AY186620. The clone was subcloned into the expression vector pMET琢-B. The recombinant plasmid was trans- formed into the pMAD16. The results of SDSanalysis indicated that expressed in the receptor and the AGPase activities of induced recombinant yeast were increased significantly. potato cloning; yeast expression; AGPase activity 马铃薯总产和栽培面积仅次于小麦、水稻和玉 物质主要是淀粉,约占块茎干物重的 70%~80%。马 米的第 4大粮食作物Jackson, 1999,其块茎的贮藏 铃薯淀粉不仅用于医药、食品和饮料生产,还可以用 *基金项目:国家自然科学基金项目No国家高技术研究与发展计划(863)项目No.2002AA241181和湖北省科技攻关项目No. 2002001006资助。 宋波涛:男, 1975年生,华中农业大学园艺林学学院博士研究生。 **通讯作者。Author for correspondence. E-mail: potato@//0.. 收稿日期: 2004-09-20 接受日期:2004-11-10 PDF 文件使用 pdfFactory Pro 试用版本创建 //.第 3期 宋波涛等:马铃薯腺苷二磷酸葡萄糖焦磷酸化酶小亚基基因 的克隆及其在毕赤酵母中的表达 283 ,因而淀粉合成与 作造纸、包装、纺织和化工原料等 温度20依1℃条件下培养 60d,以成苗并形成试管 调控是现代农业的主要研究领域之一。而腺苷二磷 块茎。将试管块茎收获后,于 4℃贮藏 7d进行低温 酸葡萄糖焦磷酸化酶 ADP- glucose pyrophosphory- 诱导处理,然后取 2g试管块茎,用 Logemann等 lase, AGPase是植物淀粉生物合成过程中的限速酶 1987 的方法抽提总 RNA。最后将 RNA溶解于 Sweetlove ., 1999,催化 G-1-P与 ATP作用,形 100滋L DEPC处理的无菌水中,电泳检测 RNA的 成 ADPG和焦磷酸 G-1-P+ATP→ADPG+PPi, 完整性,用紫外分光光度计UV-2401PC,日本岛津 ADPG则是淀粉合成的葡萄糖基供体。前人研究表 检测 RNA的浓度与纯度。 明, AGPase对淀粉合成的一般控制系数在 0.3~0.6 1.4 cDNA合成、 RACE扩增和全长 cDNA的获得 Sweetlove ., 1999,而且马铃薯淀粉合成的曲线 与克隆