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）克隆和测序与抗Cdc 42抗体交叉反应的玉米蛋白的cDNA; 2）系统分离拟南芥皮层微管功能缺陷突变体。Cdc 42是rho亚家族的一个小的GT酶，存在于从酵母到人类的许多真核生物中，并且已知参与细胞骨架的调节和MAP激酶级联的激活。以前，我们发现，针对人Cdc 42的C-末端寡肽的抗体识别约。50 kDa和约。25 kDa的蛋白质在玉米和烟草植物的细胞提取物中，以及标记的抗体微管结构和胞质溶胶。本文报道了利用免疫筛选法从玉米λ表达文库中克隆cDNA克隆，并对克隆进行了DNA序列测定。一个克隆的DNA序列含有编码与铜结合氧化酶家族成员同源的57 kDa蛋白质的ORF。需要进一步证实该蛋白在微管上的定位。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，因为微管相关基因的强突变等位基因预计是致命的。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体植物候选物，以及随后在23℃（拟南芥允许温度）下生长后其种子的恢复。使用这种温度循环方法作为筛选的第一步，通过显微镜观察微管进行第二步筛选。