Molecular Cloning of an a-Amylase cDNA from Germinating Cotyledons of Kidney Bean(Phaseolus vulgaris L. cv. Toramame)

Abstract

A cDNA coding a-amylase of Phaseolus vulgaris L. cv. Toramame was isolated and sequenced. PCR primers were synthesized based on the partial amino acid sequences of the purified a-amylase. The DNA fragment amplified by PCR with the primers was applied as a probe to screen the cDNA library representing mRNA in the cotyledons of germinating seeds. The cDNA sequence in the positive clone encoded a unique 1263-nucleotide open reading frame flanked by 5'- and 3'-untranslated regions of 6 and 194 nucleotides, respectively. The open reading frame encoded a protein of 420 amino acid residues including a 23 amino-acid-long signal peptide. The kidney bean a-amylase shows over 70% similarity in the amino acid sequence with other dicotyledonous plant a-amylases. Especially, 94% of the residues are identical to a-amylase from Vigna radiate. Kidney bean a-amylase also has all four sequence regions highly conserved in enzymes belonging to the amylase family.