Gene, Enzyme and Physiological Characterization of Shrunken-2 Isoalleles

Shrunken-2 等位基因的基因、酶和生理特征

• 批准号: 9982626
• 负责人: L. Curtis Hannah
• 金额: $ 34.5万
• 依托单位: University of Florida
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-04-01 至 2004-03-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9982626&HistoricalAwards=false
• 关键词: Gene; Enzyme; Physiological; Characterization; Shrunken

Starch comprises one of the major glucose polymers in all living organisms. It is essential for plant life since it provides a mechanism to store the vast amounts of fixed carbon and, as the the major component of many important seeds, starch provides the important energy sourcetransferred from parent to progeny. Animal life is also dependent on starch since animals (including humans) rely primarily on the starch-rich cereal seeds for essential calories and nutrients. Because of the importance of starch to both plant and animal life, the Hannah lab hasfocused on the enhancement of starch content in cereal seeds through genetic manipulation of the rate-limiting enzyme, ADP-glucose pyrophosphorylase (AGP). AGP catalyzes the synthesis of ADP-glucose and pyrophosphate from glucose-1-PO4 and ATP. ADP-glucose then serves as the glucose donor for the carbon found in starch. The work to be performed focuses on the allosteric properties of AGP. The triose sugar, 3-phosphoglyceric acid (3-PGA) activates and phosphate inhibits AGP. Specific aims include determination of whether the low level of 3-PGA activation of the maize endosperm AGP reflects loss of activation potential or an independence of 3-PGA for maximum activity. Other experiments include analysis of hybrid AGPs involving the potato tuber small subunit and the maize large subunit to identify sites involved in allosteric regulation and catalysis as well as determination of whether the strikingly different levels of activity seen in maize and in E. coli of an allosterically-altered maize AGP variant termed Sh2-Rev6 involves differential modification of the inserted amino acids. Amino acid insertions at this position that do not restore activity will be identified. Finally, a large scale mutagenesis project will be initiated to identify motifs involved in enzyme assembly, catalysis and allosteric regulation. The impressive field performance of Sh2-Rev6 in maize and now in another monocot as well as in a dicot points up the critical importance of the regulatory properties of AGP. The studies to be performed here should provide definitive insight into the mechanism of Sh2-Rev6 action as well as possibly identify other important variants.

淀粉是所有生物体中主要的葡萄糖聚合物之一。它是植物生命所必需的，因为它提供了一种储存大量固定碳的机制，并且作为许多重要种子的主要成分，淀粉提供了从亲本转移到后代的重要能量来源。 动物的生命也依赖于淀粉，因为动物（包括人类）主要依赖于富含淀粉的谷物种子来获得必需的热量和营养。由于淀粉对植物和动物生命的重要性，Hannah实验室专注于通过限速酶ADP-葡萄糖焦磷酸化酶（AGP）的遗传操作来提高谷物种子中的淀粉含量。 AGP催化葡萄糖-1-磷酸和ATP合成ADP-葡萄糖和焦磷酸。 然后ADP-葡萄糖作为淀粉中碳的葡萄糖供体。要进行的工作集中在AGP的变构特性。 丙糖、3-磷酸甘油酸（3-PGA）激活AGP，磷酸盐抑制AGP。 具体目的包括确定玉米胚乳AGP的低水平3-PGA活化是否反映活化潜力的丧失或3-PGA对于最大活性的独立性。 其他实验包括分析涉及马铃薯块茎小亚基和玉米大亚基的杂交AGP，以鉴定参与变构调节和催化的位点，以及确定玉米和E中是否存在显着不同的活性水平。在大肠杆菌中，变构改变的玉米AGP变体Sh 2-Rev 6涉及插入的氨基酸的差异修饰。 将鉴定在该位置不恢复活性的氨基酸插入。 最后，一个大规模的诱变项目将启动，以确定参与酶组装，催化和变构调节的基序。 Sh 2-Rev 6在玉米中以及现在在另一种单子叶植物和双子叶植物中的令人印象深刻的田间表现指出了AGP的调节特性的至关重要性。 在此进行的研究应提供对Sh 2-Rev 6作用机制的明确见解，并可能识别其他重要变体。