Biogenesis of Cellular Organelles (Peroxisomes) in Plants

这项研究计划的主要目标是产生新的 关于植物过氧化物酶体生物发生的信息， 乙醛酸酶体，其在储存脂质中起关键作用 关键异养生长阶段的代谢 经济上重要的油料种子。 主要强调的是 破译负责确定目标的机制， 将膜磷脂和酶蛋白导入 成熟和发芽的棉花种子中的乙醛酸酶体。 了解细胞器生物发生（细胞内不可或缺的 （二）最终是一个重要的组成部分。 理解细胞新陈代谢的复杂性。 将通过电子显微镜分析乙醛酸酶体的形成。 显微镜结合酶的免疫细胞化学， 在发育中的子叶的薄切片中显现图像。 细胞器膜的磷脂组成将是 通过Iatrascan TLC/FID定量，并且 磷脂合成酶将由 放射性标记测定通过密度- 梯度离心法 体内（原生质体）和体外 （Poly A+ mRNA的翻译产物）系统将被 建立的目的是通过实验评估以下方面的关键要求 将异柠檬酸裂解酶、过氧化氢酶和苹果酸合酶导入 从棉花和其他物种中分离的过氧化物酶体。 重组 启动DNA实验，其中λ gt 10文库 将进行测序，具有全长开放阅读框的 将其亚克隆到载体中，用于建立体外 转录/翻译系统设计用于评估分子 对蛋白质的输入进行修改。 的定时和 成熟种子中基因转录本的出现将是 用克隆的DNA探针测定， 成熟种子中酶合成的翻译控制。

The major goal of this research proposal is to generate new information on the biogenesis of a plant peroxisome, the glyoxysome, which is pivotally involved in storage lipid metabolism during the critical heterotrophic growth phase of economically - important oilseeds. The main emphasis is on deciphering the mechanisms responsible for targeting and importing membrane phospholipids and enzyme proteins into glyoxysomes in maturing and germinated cotton seeds. Understanding organelle biogenesis (integral to intracellular compartmentation) is an essential part of ultimately comprehending the complexities of cellular metabolism. Formation of glyoxysomes will be analyzed by electron microscopy coupled with immunocytochemistry of enzymes to visualize images in thin sections of developing cotyledons. Phospholipid composition of organelle membranes will be quantitated by Iatrascan TLC/FID, and the subcellular site of phospholipid synthesizing enzymes will be determined from radioactive labeling assays of fractions prepared by density - gradient centrifugation. In vivo (protoplasts) and in vitro (translation products from Poly A+ mRNA) systems will be established to experimentally evaluate critical requirements for import of isocitrate lyase, catalase, and malate synthase into peroxisomes isolated from cotton and other species. Recombinant DNA experiments will be initiated wherein a lambda gt10 library will be sequenced and those with full length open reading frames will be subcloned into vectors for establishing an in vitro transcription/translation system designed to assess molecular modifications on the import of proteins. The timing and appearance of gene transcripts in maturing seeds will be determined with cloned DNA probes to document apparent translation control(s) of enzyme synthesis in maturing seeds.***