Biogenesis of Cellular Organelles (Peroxisomes) in Plants

这项研究计划的主要目标是产生有关植物过氧化体的生物发生的新信息，乙氧体在重要的经济油料异养生长的关键阶段参与储藏脂肪代谢。主要的重点是破译负责靶向和将膜磷脂和酶蛋白导入成熟和萌发的棉花种子中的乙氧基体的机制。了解细胞器的生物发生(细胞内分隔所必需的)是最终了解细胞代谢的复杂性的重要部分。乙氧基体的形成将通过电子显微镜和酶的免疫细胞化学相结合的方法进行分析，以便在发育中的子叶的薄片中显示图像。利用Iatrascan TLC/FID对细胞膜磷脂组成进行定量，通过密度梯度离心法制备的组分进行放射性标记分析，确定磷脂合成酶的亚细胞位置。将建立体内(原生质体)和体外(Poly A+mRNA翻译产物)系统，以实验评估将异柠檬酸裂解酶、过氧化氢酶和苹果酸合酶引入棉花和其他物种的过氧化物体的关键要求。将启动重组DNA实验，其中将对lambda gt10文库进行测序，并将具有全长开放阅读框架的文库亚克隆到载体中，以建立一个体外转录/翻译系统，旨在评估蛋白质进口时的分子修饰。成熟种子中基因转录物的时间和外观将用克隆的dna探针来确定，以证明成熟种子中酶合成的表观翻译控制(S)。

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.***