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

本研究旨在1)克隆和测序与抗cdc42抗体交叉反应的玉米蛋白的cdna； 2)系统分离皮层微管功能缺陷突变体。Cdc42是rho亚家族的一个小GTPase，存在于从酵母到人类的许多真核生物中，已知参与细胞骨架的调节和MAP激酶级联的激活。先前我们发现，针对人Cdc42 c端寡肽的抗体识别玉米和烟草细胞提取物中的ca.50 kDa和ca.25 kDa蛋白，并标记微管结构和细胞质。本文报道了利用玉米cDNA λ表达文库的免疫筛选和cDNA克隆的DNA测序。其中一个克隆的DNA序列包含一个编码57 kDa蛋白的ORF，该蛋白与铜结合氧化酶家族成员同源。需要进一步确认该蛋白在微管上的定位。在第二个项目中，我们首先尝试建立有效的筛选温度敏感突变体的方法，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候选突变植株，以及它们在23℃（拟南芥的允许温度）下生长后的种子恢复情况。以该温度循环法作为筛选的第一步，进行微管微观观察筛选的第二步。

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.