PCR Cloning and Sequencing of the β-Amylase cDNA from Barley

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Abstract
Polymerase chain reaction (PCR) amplification of mRNA from developing barley (cultivar Haruna two-rows) endosperm was used to clone and sequence full-length cDNA encoding β-amylase. The β-amylase cDNA was 1, 775 bp in length. The β-amylase was deduced to be composed of 535 amino acid residues and its molecular weight was calculated to be 59, 610. Kreis et al reported that the β-amylase cDNA from barley (cultivar Hiproly) was 1, 754 bp in length and coded for a polypeptide of 535 amino acids [Eur. J. Biochem. (1987) 169, 517–525], A comparison of the β-amylase sequences from Haruna two-rows and Hiproly barleys revealed nine differences in the nucleotide sequence which resulted in three changes in the amino acid residues and 21 additional nucleotides at its 3'-end in the cultivar Haruna two-rows. The three changes were as follows: Ala-233, Ser-347, Met-527 (Haruna two-rows) and Val-233, Met-347, Ile-527 (Hiproly). Lundgard and Svensson pointed out that 23 amino acid residues of the peptide fragment derived from the COOH-terminal region of barley (cultivar Gula) β-amylase were in agreement with the deduced amino acid sequence reported by Kreis et al., with the exception of a single position (Met-527 compared to Ile) [ Carlsberg Res. Commun. (1986) 51, 487–491]. Our findings described above showed Met-527 is reasonable. In the cases of β-amylases from soybean and sweet potato, the positions that corresponded to those at 233 and 347 in the amino acid sequence of β-amylase from barley were Ala and Ser, respectively. Therefore, Ala-233 and Ser-347 in the amino acid sequence of barley β-amylase were thought to be reasonable. Sequence homology of barley β-amylase with the enzymes from soybean and sweet potato amounted to 66.7 and 59.2%, respectively.

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