Biogenesis of Cellular Organelles (Peroxisomes) in Plants

本研究计划的主要目的是为植物过氧化物酶体（glyoxysome）的生物发生提供新的信息，该酶体在具有重要经济意义的油籽异养生长的关键阶段至关重要地参与了储存脂质代谢。主要的重点是破译在成熟和发芽的棉花种子中，膜磷脂和酶蛋白靶向和导入乙氧基体的机制。了解细胞器生物发生（细胞内区隔的组成部分）是最终理解细胞代谢复杂性的重要组成部分。将通过电子显微镜结合酶的免疫细胞化学在发育中的子叶薄片上可视化图像来分析乙醛酶体的形成。细胞器膜的磷脂组成将通过Iatrascan TLC/FID进行定量，磷脂合成酶的亚细胞位点将通过密度梯度离心制备的组分的放射性标记测定。将在体内（原生质体）和体外（Poly A+ mRNA的翻译产物）建立系统，以实验评估从棉花和其他物种分离的过氧化物酶体中导入异柠檬酸裂解酶、过氧化氢酶和苹果酸合成酶的关键要求。重组DNA实验将启动，其中lambda gt10文库将被测序，具有全长开放阅读框的文库将被亚克隆到载体中，用于建立体外转录/翻译系统，旨在评估蛋白质进口的分子修饰。成熟种子中基因转录物的时间和外观将通过克隆DNA探针确定，以记录成熟种子中酶合成的明显翻译控制。***

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.***