Isolation of cytosolic ascorbate peroxidase gene and its expression in strawberry (Fragaria x ananassa Duch.)

Abstract

Strawberry (Fragaria x ananassa Duch.) fruit originates from and is composed of receptacle and achene, which is a unique type differently from the other plant fruits. Although the major part of fruit is fruit-like tissue, it is known that their development is regulated in similar ways to genuine fruit. The complex physiological and biochemical changes in fruit in distinction from other tissues result, at least in part, from changes in the amounts or activities of various enzymes and other proteins present in the fruit tissue. Four fruit-specific and -dominant proteins (10, 26, 40, 60 KDa proteins) were isolated and determined their N-terminal amino acid sequences. Three proteins with molecular weights of 10, 40, 60 KDa were presumed as proteins with the masked N-teminal. N-terminal amino acid sequence of a protein with molecular weight of 26 KDa showed the significant similarity with plant ascorbate peroxidase (APX) and was found to show APX activity. The results suggested that APX has any function in relation to fruit-specificity. As the first step to characterize 26KDa protein in molecular level, cDNA encoding the protein was isolated from strawberry fruit. Using degenerate oligonucleotides corresponding to a part of the N-terminal sequence and three sequences corresponding to conserved regions in previously reported plant APX, two RT-PCR products of 730 bp and 630 bp were synthesized. Screening of a strawberry fruit cDNA library with the PCR-amplified DNA fragments as probes resulted in cDNAs that encodes a cytosolic APX (cAPX) isoform. DNA sequenceing revealed that cDNAs contain an open reading frame of 250 amino acids (27.3kDa). The deduced amino acid sequence showed significant homology of about 90% identity with other plant cAPX. The catalytic triad and other functional site were conserved. The lack of a transit peptide in the deduced amino acid sequence implies that it encodes a cytosolic isoform. RNA blot analysis showed that the mRNA is strongly exp...