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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：
09660114
负责人：
MAESHIMA Masayoshi
金额：
$ 2.11万
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：
1997
资助国家：
日本
起止时间：
1997 至 1998
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09660114/
关键词：
Vasuolar membrane Plasma membrane Aquaporin Molecular diversity Green fluorescent protein GFP 細胞伸長 cDNAクローニング

项目摘要

Plant cells contain two different types of water channel (aquaporin) ; namely, plasma- membrane and vacuolar-membrane types. We cloned two cDNAs for the vacuolar membrane aquaporins (gamma- and delta-VM23) and six cDNAs for the plasma membrane aquaporins (PAQ1, 1b, 1c, 2, 2b, and 2c). The plasma membrane aquaporins (PAQ) contain long hydrophilic sequences at the N-terminal parts and the calculated molecular masses are larger than that or vacuolar membrane aquaporins (VM23). On SDS-PAGE the VM23 and PAQ proteins gave protein bands of 23 and 28 kDa, respectively. The transcription level of each aquaporin isoforms in the different tissues of radish were determined by northern hybridization with cDNA probes. The mRNA levels of all aquaporins were high in the seedling hypocotyls and growing taproots. In these tissues, a large amount of aquaporins may be required for cell expansion and rapid water transport across the large tissues. We also observed the differences in the transcriptional levels among tissues. For example, mRNAs of gamma- and delta-VM23 were scarcely detected in the mature leaves, leaf veins and petioles, and the level of PAQ2 mRNA was high in flower buds. In conclusion, the gene expression of each aquaporin was regulated independently, but in the growing tissues and taproots all aquaporins were coordinately expressed to provide a large amount of aquaporins. The amount of aquaporins in each tissue was also determined by immunoblot analysis with isoform-specific antibodies. Furthermore, we succeeded in expression of fusion proteins of aquaporin (gamma-VM23 and PAQ1) with green fluorescent protein in tobacco BY-2 cells. We plan to analyze the signal sequence in aquaporins to localize on the plasma and vacuolar membranes and the mechanism of vacuole biogenesis.

植物细胞含有两种不同类型的水通道（水通道蛋白）;即质膜和液泡膜类型。我们克隆了两个液泡膜水通道蛋白（γ-和δ-VM 23）的cDNA和六个质膜水通道蛋白（PAQ 1，1b，1c，2，2b和2c）的cDNA。质膜水通道蛋白（PAQ）在N端含有较长的亲水性序列，其分子量大于液泡膜水通道蛋白（VM 23）。在SDS-PAGE上，VM 23和PAQ蛋白分别给出23和28 kDa的蛋白条带。利用cDNA探针进行北方杂交，测定了水通道蛋白各亚型在萝卜不同组织中的转录水平。所有水通道蛋白的mRNA水平在幼苗下胚轴和生长的主根中都很高。在这些组织中，可能需要大量的水通道蛋白用于细胞扩增和跨大组织的快速水运输。我们还观察到不同组织间转录水平的差异。例如，γ-和δ-VM 23的mRNA在成熟叶片、叶脉和叶柄中几乎检测不到，而PAQ 2的mRNA在花芽中的水平较高。综上所述，每个水孔蛋白的基因表达是独立调控的，但在生长组织和主根中，所有水孔蛋白协调表达，提供大量的水孔蛋白。每个组织中的水通道蛋白的量也通过用同种型特异性抗体的免疫印迹分析来确定。此外，我们还成功地在烟草BY-2细胞中表达了水通道蛋白（gamma-VM 23和PAQ 1）与绿色荧光蛋白的融合蛋白。我们计划分析水通道蛋白中的信号序列，定位于质膜和液泡膜上，以及液泡生物发生的机制。