|Budget Amount *help
¥3,300,000 (Direct Cost : ¥3,300,000)
Fiscal Year 1998 : ¥1,200,000 (Direct Cost : ¥1,200,000)
Fiscal Year 1997 : ¥2,100,000 (Direct Cost : ¥2,100,000)
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 lev
els 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.
本研究では、水チャネルの細胞膜、液胞膜への局在化機構を解明することを目的に、green fluorescent protein(GFP)と水チャネル分子の融合タンパク質をタバコ培養細胞BY-2で発現させることを試みた。いずれの水チャネル分子も目的の膜に局在することを確認できた。本研究により、水チャネル分子を通して植物の成長、組織分化、水生理現象を、今後さらに解明していくための基礎的知見と実験系が整った。 Less