MOLECULAR GENETICS OF HMG-COA REDUCTASE IN ARABIDOPSIS

我们的长期目标是阐明控制基因的机制 在植物发育过程中的表达。作为了解的第一步 在此基础上，我们建议研究类异戊二烯的生物合成 通过利用我们的能力用生化分离突变体 并获得这些活性的克隆基因。 在植物中，广泛的异戊二烯类化合物，包括激素， 甾醇和光合色素，在新陈代谢和 发展。所有的类异戊二烯都来自一种共同的前体， 甲伐他酸(MVA)，由HMG辅酶A还原酶合成。这 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。我们的直接客户 目的：阐明HMG辅酶A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 使用分子探针进行还原酶检测。HMG辅酶A的两个结构基因 在拟南芥中已鉴定出还原酶和同工酶专一性 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Northern研究 和Western印迹分析及原位杂交检测植物对 环境变化、新陈代谢反馈和发育线索。在……里面 此外，将使用抗还原酶抗体来确定 两种同工酶在细胞内的分离和定位 免疫定位。 作为补充，我们设计了一些方案来隔离 利用常规和分子手段获得拟南芥中的类异戊二烯突变体 遗传学：(1)有条件和无条件的MVA营养缺陷症 在突变植物种群中根据营养成分进行鉴定 要求MVA，(2)反义RNA将用于抑制HMG CoA 还原酶的表达，(3)突变后的植物还原酶基因将被 最初在酵母中鉴定，随后转化为 为测试拟南芥，(4)将试图扰乱 利用新兴基因打靶技术研究HMG辅酶A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，并提供了遗传基础 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.