Characterization of Ubiquitin Pathway in Higher Plants

9306759 Callis真核泛素途径涉及蛋白质泛素与细胞蛋白质的共价结合。泛素化的蛋白质可以被作为蛋白质降解的靶点，或者泛素化可以导致稳定的翻译后修饰。因此，泛素途径是真核生物中调节蛋白质水平及其活性的重要途径。泛素修饰底物的分离和鉴定对于理解该途径的调节和特异性是至关重要的。虽然根据免疫印迹研究泛素化蛋白的数量似乎很多，但在体内识别的底物数量很少。在高等植物中，只有一种内源植物蛋白被鉴定为泛素途径的底物。两种不同的方法，以拟南芥为模型系统，在体内鉴定和分离高等植物的底物，将被详细概述和执行。体内其他底物的鉴定将进一步加深我们对UB途径在高等植物中的作用的了解，并有助于理解泛素化的调节。泛素途径的两个组成部分将被克隆，它们在高等植物中相对不具特征，对于理解该途径的调控是重要的。它们的等位作用也将有助于实现一个长期目标，即产生与体内发现的特异性相同的体外结合系统。在高等植物中，泛素由一个多基因家族编码。拟南芥泛素基因家族的所有成员都已被分离。多个泛素基因的作用将通过环境和发育变化的泛素基因表达的研究来解决。此外，还将测定泛素基因在其他生态型中的表达，以帮助识别功能重要的基因。这两种方法的结合将增加我们对泛素途径在高等植物代谢、发育和生理中的理解，并确定其在植物对环境胁迫的反应中的作用。***

9306759 Callis The eukaryotic ubiquitin pathway involves the covalent attachment of the protein ubiquitin to cellular proteins. Ubiquitinated proteins can then be targeted for proteolysis, or ubiquitination can result in a stable post-translational modification. Therefore the ubiquitin pathway represents an important pathway regulating protein levels and their activities in eukaryotes. The isolation and identification of substrates for ubiquitin modification are essential for an understanding of the regulation and specificity of this pathway. While the number of ubiquitinated proteins appears to be large based on immunoblot studies, the number of identified in vivo substrates is small. In higher plants, only one endogenous plant protein has been identified as a substrate of the ubiquitin pathway. Two different approaches towards identifying and isolating higher plant in vivo substrates using Arabidopsis thaliana as the model system are outlined in detail and will be performed. Identification of additional in vivo substrates will further our knowledge of the roles of the ub pathway in higher plants and contribute toward an understanding of the regulation of ubiquitination. Two components of the ubiquitin pathway that are relatively uncharacterized in higher plants and are important for an understanding of the regulation of the pathway will be cloned. Their isoation will also contribute toward a long term goal of producing an in vitro conjugation system with specificity identical to that found in vivo. Ubiquitin is encoded by a multi-gene family in higher plants. All members of the ubiquitin gene family in Arabidopsis thaliana have been isolated. The roles of multiple ubiquitin genes will be addressed through studies of the environmental and developmental changes in ubiquitin gene expression. In addition, the expression of ubiquitin genes in other ecotypes will be determined to assist in the identification of functionally important genes. A combination of these two approaches will increase our understanding of the ubiquitin pathway in higher plant metabolism, development, and physiology, and define its role in mediating plant responses to environmental stresses. ***