Signal transduction pathway controlling the contical microtubules in higher plants : Investigation about the involvement of Cdc42 GTPase-like proteins and preliminary study for the isolation of microtubule mutants

高等植物控制圆锥形微管的信号转导通路：Cdc42 GTPase 样蛋白参与的研究及微管突变体分离的初步研究

• 批准号: 10640651
• 负责人: SAKAGUCHI Shuichi
• 金额: $ 2.11万
• 依托单位: Nara Women's University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1998
• 资助国家: 日本
• 起止时间: 1998 至 1999
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10640651/
• 关键词: Arabidopsis thaliana; copper-binding oxidase; cytoskeleton; gene cloning; GTP-binding protein; microtubule; Zea mays; temperature-sensitive mutant; CDC42; 細胞質表層微小管; 情報伝達; 突然変異体

This study aimed at 1) cloning and sequencing of cDNAs for the maize proteins that cross-react with anti-Cdc42 antibody and 2) systematic isolation of Arabidopsis mutants defective in cortical microtubule function. Cdc42 is a small GTPase of the rho sub-family, found in many eukaryotes from yeast to human, and is known to be involved in the regulation of cytoskeleton and the activation of MAP kinase cascade. Previously we found that the antibody directed for C-terminal oligo-peptide of human Cdc42 recognized ca.50 kDa and ca.25 kDa proteins in cell extracts of maize and tobacco plants, and the antibody labeled microtubular structures as well as cytosol. Here, we report the cloning of cDNA clones by immunoscreening of λ expression library of maize cDNA and the DNA sequencing of the clones. The DNA sequence of one clone contained an ORF coding for a 57 kDa protein with a homology to members of the copper-binding oxidase family. It is further needed to confirm this protein's localization on microtubules. In the second project, we first tried to establish the efficient method for screening of temperature-sensitive mutants, because strong mutant alleles of microtubule-related genes are expected to be lethal. By cycling the culture temperature, 15-23-31-23℃, we could observe many candidates for mutant plants with various morphological abnormalities, as well as subsequent recovery of their seeds after the growth at 23℃, a Arabidopsis permissive temperature. Using this temperature-cycling method as the first step of screening, the second step screening by microscopic observation of microtubules will be carried out.

本研究旨在 1) 与抗 Cdc42 抗体发生交叉反应的玉米蛋白 cDNA 的克隆和测序，以及 2) 系统分离皮质微管功能缺陷的拟南芥突变体，Cdc42 是 rho 亚家族的一种小型 GTP 酶。存在于从酵母到人类的许多真核生物中，已知其参与细胞骨架的调节和 MAP 激酶级联的激活。发现针对人 Cdc42 C 端寡肽的抗体可识别玉米和烟草植物细胞提取物中的 ca.50 kDa 和 ca.25 kDa 蛋白质，并且该抗体标记了微管结构以及细胞质。通过免疫筛选玉米 cDNA 的 λ 表达文库来克隆 cDNA 克隆，并对克隆进行 DNA 测序。一个克隆的 DNA 序列包含编​​码 57 kDa 蛋白质的 ORF，其具有需要确认该蛋白在微管上的同源性。在第二个项目中，我们首先尝试建立筛选温度敏感突变体的有效方法，因为其他微管的强突变等位基因。相关基因预计是致命的。通过循环培养温度，15-23-31-23℃，我们可以观察到许多具有各种形态异常的突变植物候选者，以及随后的恢复。种子在拟南芥允许温度23℃下生长后，以这种温度循环方法作为第一步筛选，通过微管显微镜观察进行第二步筛选。