Molecular cloning of a cDNA encoding tonoplast water channel of pear fruitand its expression during development

Cloning a cDNA encoding tonoplast water channel of pear fruit disclosed tha t its deduced amino acid sequence has 77%, 52%, and 50% identities to gamma tonoplast intrinsic protein(TIP), delta TIP, and a TIP or Arabidopsis, res pectively. Therefore, this clone is considered to be a member of gamma TIPs and is designated as "Py- gamma TIP". Deduced amino acid sequence of Py- g amma TIP has six membrane spanning domains and two NPA motifs, which are co nserved within all water channels. Antibody against VM23, which is the vacu olar water channel of radish, recognized two proteins derived from tonoplas t 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- gamma TIP is 4.86; there fore, the major spot in 2D - PAGE is considered to be a translated product from Py- gamma TIP which might play a dominant role in fruit. Northern blot analysis showed that Py- gamma TIP was expressed especially high at 23 and 41 days after flowering (DAF), which correspond to the middle and end of t he cell-division stage of fruit development. This suggests that the role of Py- gamma TIP might be to sustain vacuole biogenesis and to prepare the ce lls for the rapid increase in vacuolar volume at the cell - division stage of a fruit.