MOLECULAR GENETICS OF HMG-COA REDUCTASE IN ARABIDOPSIS

拟南芥中 HMG-COA 还原酶的分子遗传学

• 批准号: 2182352
• 负责人: ROBERT M LEARNED
• 金额: $ 11.77万
• 依托单位: UNIVERSITY OF CALIFORNIA AT DAVIS
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-01-01 至 1995-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2182352
• 关键词: Arabidopsis; HMG coA reductases; SDS polyacrylamide gel electrophoresis; enzyme linked immunosorbent assay; enzyme mechanism; fusion gene; gene expression; gene mutation; genetic manipulation; genetic regulation; genetic regulatory element; genetic transcription; genetic translation; immunoprecipitation; in situ hybridization; isoprenoid; laboratory mouse; laboratory rat; molecular cloning; molecular genetics; nucleic acid probes; nucleic acid sequence; plant genetics; plant growth /development; posttranslational modifications; protein sequence; structural genes; western blottings

Our long term goal is to elucidate the mechanisms that control gene expression during plant development. As a first step toward understanding the basis for this regulation, we propose to study isoprenoid biosynthesis by taking advantage of our ability to isolate mutants with biochemical defects in the pathway and to acquire cloned genes for these activities. In plants, a wide range of isoprenoid compounds, including hormones, sterols and photosynthetic pigments, play vital roles in metabolism and development. All isoprenoids are derived from a common precursor, mevalonic acid (MVA), which is synthesized by HMG CoA reductase. This enzyme plays a key role in regulating and coordinating isoprenoid biosynthesis, but the basis of this control remains obscure. Our immediate objective is to elucidate the mechanisms of HMG CoA reductase regulation by physiological, molecular and genetic analyses. To this end, we will study the organization and regulation of HMG CoA reductase using molecular probes. Two structural genes for HMG CoA reductase have been identified in Arabidopsis and isozyme-specific antibodies will be prepared against HmG CoA reductase fusion proteins expressed in E. coli. Regulation of reductase will be examined by Northern and Western blot analysis and in situ hybridization in plants responding to environmental changes, metabolic feedback and developmental cues. In addition, anti-reductase antibodies will be used to determine the localization of the two isozymes within the cell by fractionation and immunolocalization. As a complementary approach, we have devised a number of schemes to isolate isoprenoid mutants in Arabidopsis using both conventional and molecular genetics: (1) conditional and non-conditional MVA auxotrophs will be identified in mutant plant populations on the basis of a nutritional requirement for MVA, (2) anti-sense RNA will be used to inhibit HMG CoA reductase expression, (3) mutants in the plant reductase gene will be identified initially in yeast, and subsequently transformed into Arabidopsis for testing, (4) attempts will be made to disrupt the chromosomal copies of HMG CoA reductase using emerging gene targeting technologies. The characterization of these mutants will help us elucidate the steps in the isoprenoid pathway in plants and provide a genetic basis for studying isoprenoid functions during plant development.

我们的长期目标是阐明控制基因的机制， 在植物发育过程中表达。 作为理解的第一步 为了研究这种调节的基础，我们建议研究类异戊二烯的生物合成 通过利用我们分离突变体的能力， 缺陷的途径，并获得克隆的基因，这些活动。 在植物中，广泛的类异戊二烯化合物，包括激素， 甾醇和光合色素在新陈代谢中起着重要作用， 发展 所有的类异戊二烯都衍生自共同的前体， 甲羟戊酸（MVA），其由HMG CoA还原酶合成。 这 酶在调节和协调类异戊二烯中起关键作用 生物合成，但这种控制的基础仍然模糊。 我们眼前的 目的是阐明HMG CoA还原酶的调节机制， 生理、分子和遗传分析。 为此，我们将研究HMG CoA的组织和调控 还原酶的研究。 HMG CoA的两个结构基因 在拟南芥中已经鉴定了还原酶， 将制备针对HmG CoA还原酶融合蛋白的抗体 在大肠杆菌中表达杆菌 还原酶的调节将由北方 和蛋白质印迹分析和原位杂交在植物响应 环境变化、代谢反馈和发育线索。 在 此外，抗还原酶抗体将用于确定 通过分级分离在细胞内定位两种同工酶， 免疫定位 作为一种补充方法，我们设计了一些方案， 利用常规和分子生物学方法研究拟南芥类异戊二烯突变体 遗传学：（1）条件和非条件MVA营养缺陷型将 在营养的基础上， （2）反义RNA将用于抑制HMG CoA （3）植物还原酶基因中的突变体将被 最初在酵母中鉴定，随后转化为 （4）将尝试破坏拟南芥中的 使用新兴基因靶向的HMG CoA还原酶的染色体拷贝 技术. 这些突变体的特征将有助于我们阐明 植物中类异戊二烯途径的步骤，并提供遗传基础 用于研究植物发育过程中类异戊二烯的功能。