RUI: Regulation of the Pyrimidine Pathway in Plants

RUI：植物中嘧啶途径的调节

• 批准号: 9604282
• 负责人: Robert Slocum
• 金额: $ 26.49万
• 依托单位: Goucher College
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-07-01 至 2001-06-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9604282&HistoricalAwards=false
• 关键词: RUI; Regulation; Pyrimidine; Pathway; Plants

SLOCUM Pyrimidine nucleotides are precursors of DNA and RNA biosynthesis and are constituents of activated intermediates in many metabolic processes which are fundamental to growth and development in plants and all other living organisms. The basic biochemical steps in de novo pyrimidine synthesis have been conserved in prokaryotes and eukaryotes but regulation of the pyrimidine pathway is accomplished by several different strategies involving subcellular compartmentation of enzymes and metabolites, gene duplication and gene fusion, and different metabolic control mechanisms. In plants, little is known about the mechanisms which regulate pyrimidine biosynthesis, but most evidence suggests that the important sites of regulation are the first and/or second steps in the pathway, involving the enzymes carbamoylphosphate synthetase (CPSase) and aspartate transcarbamoylase (ATCase). This research will focus on the elucidation of various molecular mechanisms regulating the activity of ATCase and CPSase in plants. As a first step toward this goal, cDNAs encoding three different ATCases in pea (pyrB1, pyrB2 and pyrB3) have been cloned. The pyrB1 and pyrB2 genes have been shown to be differentially expressed in pea tissues. These genes encode ATCase proteins whose primary sequences differ by 25% and it is likely that they have very different kinetic properties. and may participate to different degrees in the regulation of pyrimidine pathway activities. This possibility is being investigated, using a variety of approaches which includes the following: 1) kinetic characterization of the pyrB1 and pyrB2 products expressed in an ATCase-deficient strain of E. coli, 2) use of nucleic acid and antibody probes to investigate expression of the genes encoding ATCase in different plant tissues and in response to various treatments that would be expected to impact pyrimidine biosynthesis. Recently, the first plant genes encoding CPSase in Arabidopsis were cloned by the investigators. Availabili ty of cDNAs encoding the small and large subunit proteins of this plant CPSase (carA and carB genes, respectively) will permit the characterization of this gene and its expression in plant tissues. Expression of these CPSase proteins in appropriate E. coli CPSase mutants will facilitate biochemical and kinetic characterizations of the plant enzyme. Overexpression of the Arabidopsis CPSase proteins in bacteria will also facilitate their purification for biochemical studies and the production of CPSase antisera. These antisera, and the cDNAs encoding the plant CPSase proteins, will be invaluable tools in investigations of the regulation of CPSase and pyrimidine pathway activities. The investigators have now established that the basic genetic organization of the pyrimidine pathway in plants more closely resembles that of bacteria than other eukaryotes. Nonetheless, plants remain the only major group of organisms in which the fundamental control mechanisms regulating de novo pyrimidine synthesis have not been elucidated. Issues such as subcellular compartmentation of ATCase and CPSase activities, the kinetic and regulatory properties of the ATCases and CPSases themselves, metabolic interactions and regulation of pyrimidine and arginine pathway activities and many other questions will be addressed in this study. Studies such as these contribute to a broader understanding of plant primary and secondary metabolism and suggest strategies for the manipulation of plant biochemistry for many practical purposes, including the development of herbicides or other compounds that modify plant growth or development, and production of plant-derived pharmaceuticals and other useful metabolites. Apart from any purely scientific advances resulting from these studies, several undergraduate students will participate in the research. These students will gain valuable training in a modern research laboratory which will help to prepare them for post-baccalaureate careers in science.

Slocum嘧啶核苷酸是DNA和RNA生物合成的前体，是许多代谢过程中活性中间体的组成部分，这些代谢过程对植物和所有其他生物的生长和发育至关重要。在原核生物和真核生物中，从头合成嘧啶的基本生化步骤是保守的，但对嘧啶途径的调控是通过几种不同的策略来完成的，包括酶和代谢产物的亚细胞区隔、基因复制和基因融合以及不同的代谢调控机制。在植物中，对嘧啶生物合成的调控机制知之甚少，但大多数证据表明，重要的调控位点是该途径的第一步和/或第二步，涉及氨基甲酰磷酸合成酶(CPSase)和天冬氨酸转氨酰基酶(ATCase)。本研究将致力于阐明调控植物ATCase和CPSase活性的各种分子机制。作为实现这一目标的第一步，已经克隆了豌豆中编码三种不同ATCase(pyrB1、pyrB2和pyrB3)的cDNA。已有研究表明，在豌豆组织中，pyrB1和pyrB2基因存在差异表达。这些基因编码ATCase蛋白，其初级序列相差25%，它们可能具有非常不同的动力学特性。并可能不同程度地参与了嘧啶途径活性的调节。目前正在利用多种方法来研究这种可能性，这些方法包括：1)在一株ATCase缺失的大肠杆菌中表达的pyrB1和pyrB2产物的动力学特征，2)使用核酸和抗体探针来研究编码ATCase的基因在不同植物组织中的表达，以及对可能影响嘧啶生物合成的各种处理的响应。最近，研究人员首次克隆了拟南芥中编码CPSase的植物基因。编码该植物CPSase的大亚基和小亚基蛋白的cDNA(分别为CarA和CarB基因)的可用性将使该基因的特征及其在植物组织中的表达成为可能。在合适的大肠杆菌CPSase突变体中表达这些CPSase蛋白将有助于植物酶的生化和动力学特性的研究。拟南芥CPSase蛋白在细菌中的过表达也有利于其纯化，为生化研究和生产CPSase抗血清奠定了基础。这些抗血清和编码植物CPSase蛋白的cDNA将是研究CPSase和嘧啶途径活性调节的宝贵工具。研究人员现在已经确定，与其他真核生物相比，植物中嘧啶途径的基本遗传组织更类似于细菌。尽管如此，植物仍然是唯一一个调节从头合成嘧啶的基本控制机制尚未阐明的主要生物群。诸如ATCase和CPSase活性的亚细胞划分、ATCase和CPSase自身的动力学和调节特性、代谢相互作用以及对嘧啶和精氨酸途径活性的调控等许多问题都将在本研究中得到解决。这样的研究有助于更广泛地了解植物的初级和次生代谢，并为出于许多实际目的而操纵植物生物化学提出了战略建议，包括开发用于改变植物生长或发育的除草剂或其他化合物，以及生产植物衍生药物和其他有用的代谢物。除了这些研究带来的任何纯粹的科学进步外，几名本科生将参与这项研究。这些学生将在一个现代化的研究实验室接受宝贵的培训，这将有助于他们为毕业后在科学领域的职业生涯做好准备。