Characterization of a plant gene encoding a small GTP-binding protein that is specifically induced by SO_2 and NO_2

与ras相关的小GTP结合蛋白参与了多种细胞过程的调控，包括生长、分化、细胞骨架组织、细胞内囊泡运输和分泌。这些基因在植物中已被发现，但对它们的调控机制和编码蛋白的功能知之甚少。小麦wgp7基因编码一个小GTP结合蛋白，由于其转录水平受SO_2和NO_2的影响而被首次鉴定。在过去的三年中，我们获得了以下结果：1.wgp7转录不仅受SO_2和NO_2的光诱导，而且依赖于光但也受到特定诱导氧化胁迫的条件，包括除草剂甲基紫精(百草枯)，过氧化氢酶抑制剂，以及暴露在寒冷或UV-C2下。在CaMV35S启动子…的控制下，转基因烟草植株(包括T2和T3代)以有意义的方向产生wgp7基因根据转基因表达水平的不同，获得了不同形态的植物，包括严重的莲座状矮生植物。在这些植物中表达了高水平的wgp7转录本和蛋白，但尚未鉴定出对几种氧化胁迫条件反应的明显差异。3.wgp7的启动子区域已被克隆并部分测序。目前的研究旨在鉴定氧化胁迫调控表达所必需的顺式元件。4.体外合成的重组wgp7蛋白，对转基因烟草的亚细胞分离表明wgp7蛋白定位于微粒体部分，提示其为膜结合蛋白。在小麦中，wgp7蛋白在氧化胁迫处理后迅速被诱导。氧化胁迫后的亚细胞定位实验正在进行中。5.利用wgp7蛋白和另一种小的GTP结合蛋白，通过相互作用克隆分离相互作用蛋白。结果，我们鉴定了一个编码新蛋白的cDNA克隆，编码一种新的蛋白对该蛋白的缺失分析表明，198个氨基酸的N末端特异区足以与该小GTP结合蛋白结合。较少

The ras-related small GTP-binding proteins have been implicated in the regulation of various cellular processes including growth, differentiation, cytoskeletal organization, intracellular vesicle transport and secretion.Such genes have been identified in plants but little is known of the mechanisms by which they are regulated or the functins of their encoded proteins.The wheat gene, wgp7, encoding a small GTP-binding protein, was first identified because of its increased transcript levels by SO_2 and NO_2.Over the last 3 years, the following results have been obtained.1.The wgp7 transcripts are rapidly induced not only by SO_2 and NO_2, in a light-dependant manner, but also by conditions that specifically induce oxidative stress, including the herbicide methyl viologen (paraquat), a catalase inhibitor, and by exposure to cold or UV-C.2.Transgenic tobacco plants (including T2 and T3 generations) were produced with the wgp7 gene in a sense orientation under control of the CaMV 35S promot … More er.Depending on the level of transgene expression, plants with differing morphologies were obtained, including a severe rosette dwarf.High levels of wgp7 transcripts and proteins were expressed in these plants, but no clear differences in response to several oxidative stress conditions have yet been identified.3.The promoter region of wgp7 has been cloned, and partially sequenced.Current studies are aimed to identify the cis-elements essential for oxidative stress regulated expression.4.Rocombinant wgp7 protein, synthesized in vitro, was shown to bind GTP.The full length and also highly-variable C-terminal end of the protein were used to raise polyclonal antibodies.Subcellular fractionation of proteins from transgenic tobacco showed that the wgp7 protein was localized to the microsomal fraction, suggesting that it is membrane-bound.In wheat, the wgp7 protein was rapidly induced by oxidative stress treatment.Experiments for subcellular localization of the protein following oxidative stress are underway.5.Using the wgp7 protein, as well as another small GTP-binding protein, interaction cloning was used to isolate interacting proteins.Consequently, a cDNA clone, encoding a novel protein was identified.Binding of this protein was found to be specific, reversible and priferentially with the small GTP-binding protein in its GDP-bound form.Deletion analysis of this protein showed that an N-terminal specific region of 198 amino acids is sufficient for binding the small GTP-binding protein. Less