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Molecular porperties and cellular function of aquaporins in the plasam and vacuolar membranes of plant

植物质体和液泡膜中水通道蛋白的分子特性和细胞功能

基本信息

批准号：
11660084
负责人：
MAESHIMA Masayoshi
金额：
$ 2.3万
依托单位：
Nagoya University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11660084/
关键词：
Vacuolar membrane Plasma membrane Aquaporin Molecular diversity Green fluorescent protein CAM plant

项目摘要

Plant aquaporins (water channels) occur in multiple isoforms and are distributed in both plasma membrane and tonoplast. We have cloned several isoforms in plasma membranes (PAQ1, 1b, 1c, 2, 2b, and 2c) and vacuolar membranes (gamma- and delta-VM23) of radish. In this study, we investigated the time- and cell-specificity of gene expression of these aquaporins, the relationship between the aquaporin content in the membrane and membrane water permeability, and mechanism of membrane localization of each aquaporin.(1) A comprehensive investigation of the aquaporin mRNAs, including VM23, in seedlings, plants, flowers and seeds of radish showed a marked accumulation of all the mRNAs in hypocotyls and growing taproots. In some tissues, the mRNA and protein levels varied with isoforms.(2) The osmotic water permeability was determined for plasma membranes and vacuolar membranes prepared from radish and Graptopetalum (a CAM plant) with a stopped flow spectrophotometer. The water permeability of the vacuolar membrane was higher than that of the plasma membrane of both plant species. Also, the permeability of both membranes prepared from CAM plant was extremely lower than that of radish membranes. The result is consistent with the abundance of aquaporin in the membrane.(3) We expressed fusion proteins of PAQ1 and gamma-VM23 with a green fluorescent protein (GFP) in tobacco cells. PAQ-GFP protein was correctly localized on the plasma membrane and VM23-GFP was on the vacuolar membrane. Deletion of N- or C-terminal part had no effect on the localization. The results indicate that the localization signal is in the internal part of aquaporins.(4) We also tried to introduce radish aquaporins (PAQ1, PAQ2, gamma-VM23 and delta-VM23) into Kalanchoe, a CAM plant, in which the aquaporin level is very low. All the genes were detected in transgenic plants and at least PAQ2 protein was found by immunoblotting. Now, we investigate physiological properties of the transgenic Kalanchoe.

植物水通道蛋白（水通道）以多种亚型存在，分布于质膜和液泡膜上。我们已经克隆了几种异构体在质膜（PAQ 1，1b，1c，2，2b和2c）和液泡膜（γ-和δ-VM 23）的萝卜。在这项研究中，我们研究了这些水通道蛋白的基因表达的时间和细胞特异性，水通道蛋白的含量和膜透性之间的关系，和膜定位的每个水通道蛋白的机制。(1)对萝卜幼苗、植株、花和种子中包括VM 23在内的水通道蛋白mRNA的全面调查显示，所有mRNA在下胚轴和生长中的主根中显著积累。在某些组织中，mRNA和蛋白质水平因亚型而异。(2)用停流分光光度计测定了萝卜和风车草（一种CAM植物）的质膜和液泡膜的渗透透性。两种植物液泡膜的透水性均高于质膜。此外，从CAM植物制备的两种膜的渗透性极低，比萝卜膜。这一结果与细胞膜上水通道蛋白的丰度一致。(3)我们在烟草细胞中表达PAQ 1和γ-VM 23与绿色荧光蛋白（GFP）的融合蛋白。PAQ-GFP蛋白正确定位于质膜上，VM 23-GFP蛋白定位于液泡膜上。N-或C-末端部分的缺失对定位没有影响。结果表明，定位信号位于水通道蛋白的内部。(4)我们还尝试将萝卜水通道蛋白（PAQ 1、PAQ 2、gamma-VM 23和delta-VM 23）导入CAM植物Kalamae中，其中水通道蛋白水平非常低。在转基因植株中检测到所有的基因，并且通过免疫印迹检测到至少PAQ 2蛋白。现在，我们研究转基因Kalabele的生理特性。