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

植物细胞含有两种不同类型的水通道(水通道蛋白)，即质膜型和液泡膜型。我们克隆了两个液泡膜水通道蛋白的cDNA(Gamma-和Delta-VM23)和六个质膜水通道蛋白的cDNA(PAQ1、1b、1c、2、2b和2c)。质膜水通道蛋白(PAQ)在N端含有较长的亲水性序列，其计算的相对分子质量大于或液泡膜水通道蛋白(VM23)。在SDS-PAGE上，VM23和PAQ蛋白分别出现23 kDa和28 kDa的蛋白条带。采用Northern杂交技术检测了萝卜不同组织中各水通道蛋白亚型的转录水平。在幼苗的下胚轴和生长的主根中，所有水通道蛋白的mRNA水平都很高。在这些组织中，可能需要大量的水通道蛋白来进行细胞扩张和水在大组织中的快速运输。我们还观察了不同组织之间转录水平的差异。例如，在成熟的叶片、叶脉和叶柄中很少检测到γ-VM23和Delta-VM23的mRNAs，而在花蕾中PAQ2的mRNA量较高。综上所述，每个水通道蛋白的基因表达是独立调控的，但在生长组织和主根中，所有的水通道蛋白都是协同表达的，以提供大量的水通道蛋白。用异构体特异性抗体进行免疫印迹分析，测定各组织中水通道蛋白的含量。此外，我们还成功地在烟草BY-2细胞中表达了水通道蛋白(γ-VM23和PAQ1)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，以定位于质膜和液泡膜上，并研究液泡生物发生的机制。

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.