Cloning and Expression of a cDNA Encoding Larval α-Amylase of Azuki Bean Weevil, <i>Callosobruchus chinensis</i>

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  • Yamada Tsuyoshi
    Laboratory of Plant Genetics and Breeding, Graduate School of Bioagricultural Sciences, Nagoya University
  • Ikeda Motoko
    Laboratory of Sericulture and Entomoresources, Graduate School of Bioagricultural Sciences, Nagoya University
  • Kobayashi Michihiro
    Laboratory of Biodynamics, Graduate School of Bioagricultural Sciences, Nagoya University
  • Hattori Kazumi
    Laboratory of Plant Genetics and Breeding, Graduate School of Bioagricultural Sciences, Nagoya University

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  • Cloning and expression of a cDNA encoding larval α-amylase of azuki bean weevil, Callosobruchus chinensis
  • Cloning and expression of a cDNA encoding larval アルファ amylase of azuki bean weevil Callosobruchus chinensis

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Abstract

α-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 α-amylase inhibitor (αAl) 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 α-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 (Glu237, Asp201, Asp303) are involved in catalysis and three histidine residues (His115, His205, His301) responsible for substrate binding conserved in this α-amylase. In baculovirus expression system, CCA expressed C. chinensis α-amylase that was active in a broad pH range from 4.5 to 6.5. Consistent with native α-amylase from C. chinensis larvae, the activity of recombinant C. chinensis α-amylase expressed in the baculovirus expression system was inhibited by αAl-1 and αAl-Pa1 from the cultivated common bean, Phaseolus vulgaris, and the tepary bean, Phaseolus acutifolius, respectively, but not by αAl-2 from certain wild common bean accessions. These results indicate that CCA and its expressed recombinant C. chinensis α-amylase obtained in this study are useful to elucidate the molecular basis for the specific interactions between bruchid α-amylases and bean αAls.

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