Targeting of Vacuolar Proteins In Plant Cells

植物细胞中液泡蛋白的靶向

• 批准号: 8817211
• 负责人: Maarten Chrispeels
• 金额: $ 55.42万
• 依托单位: University of California-San Diego
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-02-01 至 1994-01-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=8817211&HistoricalAwards=false
• 关键词: Targeting; Vacuolar; Proteins; Plant; Cells

The major objective of this work is to identify the protein domain that allows vacuolar proteins to be targeted to the vacuoles of plant cells, and to identify the receptor(s) with which they interact. The work will concentrate on three vacuolar proteins present in bean (Phaseolus vulgaris) seeds, phytohemagglutinin, phaseolin, and cx-amylase inhibitor and will use two different transgenic systems, the yeast Saccharomyces cerevisiae and tobacco (Nicotiana tabacum). In addition, we will attempt to transform bean with Agrobacterium tumefaciens to be in a position to study transport to the vacuole. The work will utilize translational fusions between portions of these three proteins and the yeast invertase gene (a secreted protein) to delineate the targeting domain. Mutagenesis will be used together with computer-based sequence comparisons to identify the critical residues. Identification of the receptor will rely on affinity studies (either columns or gel/blot binding procedures) using the targeting domains of these proteins and solubilized membrane fractions. Once the receptor has been isolated, we will attempt to clone the gene and carry out immunocytochemical work to study its subcellular distribution. This project will provide important basic information relevant to our understanding of how cellular proteins get to their specific functional sites within the cell. It will also provide important information concerning how seed storage proteins get to the sites of storage within the cell, which is of practical value in terms of genetic engineering of nutritionally "improved" seeds and grains.

这项工作的主要目标是确定蛋白质结构域， 允许液泡蛋白靶向植物细胞的液泡， 并鉴定与它们相互作用的受体。 这项工作将 主要研究菜豆（Phaseolus）中存在的三种液泡蛋白 vulgaris）种子、植物血凝素、菜豆素和CX-淀粉酶抑制剂 并将使用两种不同的转基因系统，酵母菌 酿酒酵母和烟草（Nicotiana tabacum）。 此外，我们将 尝试用根癌农杆菌（Agrobacterium tumefaciens）转化菜豆， 研究运输到液泡的位置。 这项工作将利用 这三种蛋白质的部分之间的翻译融合， 酵母转化酶基因（一种分泌蛋白）， 域 诱变将与计算机测序一起使用 比较以确定关键残留物。 识别 受体将依赖于亲和性研究（柱或凝胶/印迹 结合程序）使用这些蛋白质的靶向结构域， 溶解的膜级分。 一旦受体被分离，我们 将尝试克隆该基因并进行免疫细胞化学工作， 研究其亚细胞分布。 该项目将提供重要的基本信息， 了解细胞蛋白质如何发挥其特定功能 细胞内的位置。 它还将提供重要信息 关于种子贮藏蛋白质如何到达种子内部的贮藏位点， 细胞，这在基因工程方面具有实用价值， 营养“改良”的种子和谷物。