Molecular Dissection of the Plant Golgi: A Heterologous Approach

本项目的重点是高尔基体，它是植物细胞表面的糖蛋白和复杂多糖的生物合成、修饰和分选的亚细胞位点。人们对植物中高尔基体的分子组成知之甚少。参与细胞壁基质多糖合成的酶很少被鉴定和表征。我们对高尔基体中这些酶的组织结构一无所知，也不知道这种组织结构是如何维持的，也不知道多糖的合成是如何开始的，也不知道复杂的多糖是如何组装并靶向细胞壁上的特定位置的。部分困难在于无法将植物高尔基体分解为顺式、内式和反池式元素。本文提出了一种新的方法来实现这种分离，即利用嵌合蛋白（禽传染性支气管炎病毒、IBV-E1和GUS的E1糖蛋白之间的翻译融合）将可操纵的GUS标记靶向高尔基体。这一想法是基于对动物系统的观察，即IBV-E1被靶向并在顺式高尔基池的膜中积累。在本项目中，包含全部或部分IBV-E1编码序列的嵌合标记的超微结构位置将通过EM胶体金免疫定位（与该技术专家合作）确定。这些研究将为利用IBV-E1在植物细胞中表达的融合进行更广泛的生化工作奠定可行性。植物干重的很大一部分在细胞壁中。植物细胞壁对植物的生存至关重要，因为它们提供维持形态和膨胀压力所需的刚性。有迹象表明，细胞壁也是防御植物病原体机制的关键组成部分。它们是重要商业产品的生物来源，如纤维素和纤维素基产品、木制品和膳食纤维。这种理解植物壁生物合成的细胞生物学的新方法可能为特定目的的植物壁基因工程开辟可能性。此外，该研究还将为基因工程植物中影响动物糖蛋白表达的参数提供重要信息。这应该与涉及从转基因植物中回收商业上有用的动物糖蛋白的应用有关。

The focus of this project is on the Golgi apparatus, which is the subcellular site of the biosynthesis, modification, and sorting of glycoproteins and complex polysaccharides of the plant cell surface. Very little is known about the molecular composition of the Golgi in plants. Few of the enzymes involved in the synthesis of cell wall matrix polysaccharides have been identified and characterized. Nothing is known about the organization of these enzymes within the Golgi, or how such organization is maintained, or how polysaccharide synthesis is initiated, or how complex polysaccharides are assembled and targeted to specific locations in the cell wall. Part of the difficulty has been an inability to subfractionate plant Golgi into cis-, medial, and trans-cisternal elements. A novel approach is proposed to achieve such separation, in which a chimaeric protein (translational fusion between the E1 glycoprotein of avian Infectious Bronchitis Virus, IBV-E1, and GUS) will be used to target the manipulable GUS marker to the Golgi. This idea is based on observations in animal systems that IBV-E1 is targeted to and accumulates in the membranes of the cis-Golgi cisternae. In this project, the ultrastructural location of chimaeric markers containing all or part of the IBV-E1 coding sequence will be determined by EM colloidal gold immunolocalization (to be done in collaboration with experts in this technique). These studies will establish the feasibility of more extensive biochemical work exploiting the IBV-E1 fusion expressed in plant cells. %%% A large fraction of the dry weight of plants is in the cell walls. Plant cell walls are critical to the survival of the plants, since they provide the rigidity needed to maintain morphology and turgor pressure. There are indications that cell walls are also critical components of defense mechanisms against plant pathogens. They are the biological sources of significant commercial products, such as cellulose and cellulose-based products, wood products, and dietary fiber. This novel approach to understanding the cell biology of plant wall biosynthesis may open up the possibility of genetic engineering of plant walls for specific purposes. In addition, the research should provide important information about the parameters that affect expression of animal glycoproteins within genetically engineered plants. This should be relevant for applications involving the recovery of commercially useful animal glycoproteins from transgenic plants.