Cloning and expression of a cDNA encoding larval alpha-amylase of azuki bean weevil, Callosobruchus chinensis

摘要

alpha-Amylases are major digestive enzymes in bruchids that infest seeds of starchy grain legumes. To cope with these insect pests, some species of the genus Phaseolus have developed seed protection systems involved in various defense proteins. Proteinaceous alpha-amylase inhibitor (alpha Al) is one of these defense proteins that has been verified through genetic engineering to be a useful tool for protection of legume seeds from bruchids. In the present study, a cDNA (CCA) encoding alpha-amylase was isolated from the larvae of the azuki bean weevil, Callosobruchus chinensis. The CCA possessed an open reading frame encoding a polypeptide of 490 amino acid residues including the predicted signal sequence of 16 amino acid residues, that had an estimated molecular weight of 52,223 after cleavage of predicted signal sequence. Deduced amino acid sequence showed that three amino acid residues (Glu~(237), Asp~(201), Asp~(303)) are involved in catalysis and three histidine residues (His~(115), His~(205),His~(301)) responsible for substrate binding conserved in this alpha-amylase. In baculovirus expression system, CCA expressed C. chinensis alpha-amylase that was active in a broad pH range from 4.5 to 6.5. Consistent with native alpha-amylase from C. chinensis larvae, the activity of recombinant C. chinensis alpha-amylase expressed in the baculovirus expression system was inhibited by alpha Al-1 and alpha Al-Pa1 from the cultivated common bean, Phaseolus vulgaris, and the tepary bean, Phaseolus acutifolius, respectively, but not by alpha Al-2 from certain wild common bean accessions. These results indicate that CCA and its expressed recombinant C. chinensis alpha-amyiase obtained in this study are useful to elucidate the molecular basis for the specific interactions between bruchid alpha-amylases and bean alpha Als.