Molecular Cloning of a cDNA Encoding Tonoplast Water Channel of Pear Fruit and Its Expression during Development

Abstract

Cloning a cDNA encoding tonoplast water channel of pear fruit disclosed that its deduced amino acid sequence has 77%, 52%, and 50% identities to γ tonoplast intrinsic protein (TIP), δ TIP, and α TIP of Arabidopsis, respectively. Therefore, this clone is considered to be a member of γ TIPs and is designated as "Py-γ TIP". Deduced amino acid sequence of Py-γ TIP has six membrane spanning domains and two NPA motifs, which are conserved within all water channels. Antibody against VM23, which is the vacuolar water channel of radish, recognized two proteins derived from tonoplast proteins of pear fruit yielded a major spot at pI 4.8 and a minor one at pI 5.2 by two dimensional-polyacrylamide gel electrophoresis (2D-PAGE). Putative pI of deduced amino acid sequence of Py-γ TIP is 4.86 ; therefore, the major spot in 2D-PAGE is considered to be a translated product from Py-γ TIP which might play a dominant role in fruit. Northern blot analysis showed that Py-γ TIP was expressed especially high at 23 and 41 days after flowering (DAF), which correspond to the middle and end of the cell-division stage of fruit development. This suggests that the role of Py-γ TIP might be to sustain vacuole biogenesis and to prepare the cells for the rapid increase in vacuolar volume at the cell-division stage of a fruit.