MOLECULAR GENETICS OF HMG COA REDUCTASE IN ARABADOPSIS

我们的长期目标是阐明控制基因的机制 植物发育过程中的表达。 作为理解的第一步 在此调节的基础上，我们建议研究类异戊二烯的生物合成 利用我们用生化方法分离突变体的能力 途径的缺陷并获得这些活动的克隆基因。 在植物中，多种类异戊二烯化合物，包括激素、 甾醇和光合色素在新陈代谢和 发展。 所有类异戊二烯均源自共同的前体， 甲羟戊酸 (MVA)，由 HMG CoA 还原酶合成。 这个 酶在调节和协调类异戊二烯中起着关键作用 生物合成，但这种控制的基础仍然不清楚。 我们的即时 目的是通过以下方式阐明 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的组织和监管 使用分子探针的还原酶。 HMG CoA 的两个结构基因 已在拟南芥中鉴定出还原酶和同工酶特异性 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。 还原酶的调节将由北方研究 植物响应的蛋白质印迹分析和原位杂交 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 通过分级分离将两种同工酶定位在细胞内 免疫定位。 作为补充方法，我们设计了许多方案来隔离 使用常规和分子方法在拟南芥中类异戊二烯突变体 遗传学：（1）条件和非条件 MVA 营养缺陷型 根据营养成分在突变植物群体中鉴定 MVA的要求，（2）反义RNA将用于抑制HMG CoA 还原酶表达，（3）植物还原酶基因突变体将 最初在酵母中鉴定，随后转化为 用于测试的拟南芥，（4）将尝试破坏 使用新兴基因靶向的 HMG CoA 还原酶的染色体拷贝 技术。 这些突变体的表征将帮助我们阐明 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。

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.