Isolation of two α-amylase inhibitor genes of tepary bean (Phaseolus acutifolius A. Gray) and their functional characterization in genetically engineered azuki bean

Seeds of a tepary bean cultivar (Phaseolus acutifolius A. Gray) contain two isoforms of α-amylase inhibitor, designated as αAI-Pa1 and αAI-Pa2, that differ in their specificity for insect α-amylases. Like αAI-1 and αAI-2 of common bean (Phaseolus vulgaris L.), the active form of αAI-Pa2 is a heterotetramer of two α and two β subunits that are both generated from a common precursor, whereas αAI-Pa1 is composed of a single polypeptide. We have now isolated cDNAs encoding αAI-Pa1 and αAI-Pa2 and found that the deduced amino acid sequence αAI-Pa2 is most similar to that of αAI-2 whereas that of αAI-Pa1 most closely resembles those of seed lectins of tepary bean and common bean. Expression of these cDNA clones in azuki bean (Vigna angularis Willd. Ohwi & Ohashi) revealed that active forms of the inhibitor proteins accumulated in seeds and exhibited specificities for insect α-amylases identical to those of the native proteins purified from tepary bean seeds. The active form of αAI-Pa1 in transgenic azuki bean was a single polypeptide with a size similar to that expected for the full-length encoded protein. These results suggest that αAI-Pa1 defines a new type of bean αAI that is structurally related to lectins and is not activated by proteolytic processing.