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发育。本研究的长期目标是了解COP9信号体在植物中整合环境和发育信号的分子机制。COP9信号小体是一个8个亚基的蛋白质复合物，在这个实验室被鉴定和表征。每个亚基对应的COP/DET/FUS位点的分子克隆和鉴定已接近完成。最近在哺乳动物中发现了COP9信号体，并且可能存在于大多数多细胞生物中。最近的研究表明，COP9复合体直接与与生长素调节相关的SCF型泛素蛋白连接酶和26S蛋白酶体相互作用，因此可能在泛素-蛋白酶体途径介导的蛋白质降解调节中发挥作用。这些发现将被扩展，并在光和生长素调节的发育中研究其功能意义。本研究的具体目的是：1.研究方法：含有SCF型泛素连接酶（E3）复合物的拟南芥CUL 1与COP9信号体的体内关联将被证实，其可能受到光和其他光调节基因的调控，2。COP9信号体与生长素特异性scftir1亚型E3复合体的可能关联及其在调节生长素控制的生理反应中的可能作用将被进一步研究。酵母双杂交或体外结合试验将被用于检测COP9信号体亚基之间以及SCF复合物亚基与COP9信号体之间的成对相互作用。如果时间允许，复合体外的蛋白质包括COP1， HY5和已证实的核定位的变色色素和crytochromes也将被检查。特异性改变COP9信号体单个亚基表达或活性的转基因株系的表型将在幼苗和成年植株的形态、分子和生化水平上进行表征。利用反向遗传方法将创建弱或诱导表型，以便研究COP9和可能的亚基功能在苗期后的发育阶段。这些目标的实现将揭示进化上保守的COP9信号体的基本细胞和生化功能，以及它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.