MOLECULAR CLONING, CHARACTERIZATION AND EXPRESSION ANALYSIS OF ISOFORMS ENCODING TONOPLAST-BOUND PROTON-TRANSLOCATING INORGANIC PYROPHOSPHATASE IN TOBACCO

摘要

The tonoplast-bound proton-translocating inorganic pyrophosphatase (V-type PPase) and the proton ATPase (V-type ATPase) are electrogenic proton pumps guaranteeing the energization of solute transport across the tonoplast. Using an Arabidopsis thaliana PCR cDNA fragment corresponding to clone ATAVP3 we have isolated 24 cDNA clones encoding tonoplast-bound inorganic pyrophosphatase of tobacco. Based on restriction analysis the cDNA clones could be grouped into three different classes. The complete nucleotide sequence of one member of each class (TVP5, TVP9 and TVP31) was determined. The cDNA clones contain an uninterrupted open reading frame of 2292 bp (TVP5), 2295 bp (TVP9) and 2298 bp (TVP31) coding for polypeptides of 764, 765 and 766 amino acids, respectively. The nucleotide sequence of the different clones is highly homologous within the coding region (79-89% identity) but differs strongly in the untranslated regions. The individual classes are encoded by single- or low-copy genes as judged from genomic gel blot experiments using 3'-specific probes. RNA. analysis revealed that the accumulation of the specific transcripts is differentially regulated during leaf development.