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

编码 SO_2 和 NO_2 特异性诱导的小 GTP 结合蛋白的植物基因的表征

基本信息

批准号：
07640875
负责人：
YOUSSEFIAN Shohab
金额：
$ 1.66万
依托单位：
Biotechnology Institute, Akita Prefectural College of Agriculture
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1995
资助国家：
日本
起止时间：
1995 至 1997
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07640875/
关键词：
wheat GTP small G protein oxidative stress sulphur dioxide nitrogen dioxide transgenic plants accessory proteins Wgp

项目摘要

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

ras相关的小GTP结合蛋白参与调节细胞生长、分化、细胞骨架结构、胞内小泡运输和分泌等多种细胞过程，这些基因已在植物中被鉴定，但对其调控机制和编码蛋白功能知之甚少。小麦基因wgp 7编码一种小GTP结合蛋白，在过去的3年中，人们获得了以下结果：1. wgp 7基因的转录不仅受SO_2和NO_2的光依赖性诱导，而且受氧化应激的特异性诱导，2.在CaMV 35 S启动子的调控下，将wgp 7基因以有义方向导入烟草，获得了转基因烟草T2和T3代，并对转基因烟草的抗性进行了研究。 ...更多信息根据转基因表达的水平，获得了具有不同形态的植物，包括严重的莲座矮化。在这些植物中表达了高水平的wgp 7转录本和蛋白质，但尚未鉴定出对几种氧化胁迫条件的反应的明显差异。3.已经克隆了wgp 7的启动子区，目前的研究旨在鉴定氧化应激调节表达所必需的顺式元件。4.体外合成的重组wgp 7蛋白，结果表明，wgp 7蛋白与GTP结合，其全长和C末端高度可变，可用于制备多克隆抗体。转基因烟草的蛋白质亚细胞分级分离表明，wgp 7蛋白定位于微粒体部分，在小麦中，wgp 7蛋白被氧化胁迫处理快速诱导。氧化胁迫后该蛋白的亚细胞定位实验正在进行中。5.利用wgp 7蛋白以及另一种小GTP结合蛋白，使用相互作用克隆来分离相互作用蛋白。因此，cDNA克隆，该蛋白与小GTP结合蛋白（small GTP-binding protein，small GTP-binding protein少