Molecular Genetic Analysis of Arabidopsis COP9 Signalosome

高等植物可以监测环境信号，并以最适合当前环境的方式控制生长和发育。这项研究的长期目标是了解COP 9信号体用于整合植物环境和发育信号的分子机制。COP 9信号体是本实验室鉴定和表征的八亚基蛋白复合物。每个亚基的相应COP/DET/FUS基因座的分子克隆和鉴定已接近完成。COP 9信号体最近在哺乳动物中被发现，并且可能存在于大多数多细胞生物中。最近的工作表明，COP 9复合物直接相互作用与SCF型泛素蛋白连接酶与生长素调节和26 S蛋白酶体，从而可能发挥作用，调节蛋白质降解介导的泛素-蛋白酶体途径。这些发现将被扩展，并在光和生长素调控的发展检查其功能的影响。本研究的具体目的是：1.拟南芥CUL 1含有SCF型泛素连接酶（E3）复合物与COP 9信号体的体内结合将被证实，并将研究其可能受光和其他光调控基因的调控。COP 9信号体与生长素特异性的SCFtir 1亚型E3复合物的可能联系及其在调节生长素控制的生理反应中的可能作用将被研究.酵母双杂交或体外结合试验将组合使用，以检查COP 9信号体亚基之间以及SCF复合物亚基与COP 9信号体之间的成对相互作用。在时间允许的情况下，还将检测复合物外的蛋白质，包括COP 1、HY 5和已证实的核定位的发色团和隐色团。特异性改变COP 9信号体的单个亚基的表达或活性的转基因系的表型将在幼苗和成年植物中在形态学、分子和生物化学水平上表征。将使用反向遗传方法创建弱或诱导型表型，以研究COP 9和可能的亚基在苗期后的发育阶段中的功能。 这些目标的实现将揭示进化上保守的COP 9信号体的基本细胞和生化功能及其与调节植物生长的环境和发育信号的联系。

Higher plants can monitor environmental signals and control growth and development in a manner optimal for the prevailing environment. The long-term goal of this research is to understand the molecular mechanisms whereby the COP9 signalosome is used to integrate environmental and developmental signals in plants. The COP9 signalosome is an eight-subunit protein complex which was identified and characterized in this laboratory. Molecular cloning and identification of the corresponding COP/DET/FUS loci for each subunit is close to completion. The COP9 signalosome has recently been found in mammals and is probably present in most multicellular organisms. Recent work has suggested that the COP9 complex directly interacts with both a SCF type ubiquitin protein ligase associated with auxin regulation and the 26S proteasome and thus probably plays a role in regulated protein degradation mediated by the ubiquitin-proteasome pathway. Those findings will be extended and their functional implications in light and auxin regulated development examined. The specific aims of this research are:1. The in vivo association of an Arabidopsis CUL 1 containing SCF type ubiquitin ligase (E3) complex with COP9 signalosome will be confirmed and its possible regulation by light and other light regulatory genes investigated,2. The possible association of the COP9 signalosome with the auxin specific SCFtir1subtype E3 complex and its possible role in regulating auxin controlled physiological responses will be investigated.3. Yeast two hybrid or in vitro binding assays will be used in combination to examine the pairwise interactions among the COP9 signalosome subunits and between the subunits of SCF complex and the COP9 signalosome. The proteins outside the complexes including COP1, HY5 and the demonstrated nuclear localized phyochromes and crytochromes will also be examined as time allows.4. The phenotype of the transgenic lines which specifically alter the expression or activity of individual subunits of the COP9 signalosome will be characterized at the morphological, molecular and biochemical levels in both seedlings and adult plants. Weak or inducible phenotypes using reverse genetic approaches will be created in order to investigate both COP9 and possibly subunit function in developmental stages after the seedling stage. Accomplishment of these objectives will reveal fundamental cellular and biochemical functions of the evolutionarily conserved COP9 signalosome and its linkage with the environmental and developmental signals that regulate plant growth.