cDNA cloning, biochemical characterization and inhibition by plant inhibitors of the alpha-amylases of the Western corn rootworm, Diabrotica virgifera virgifera.
We report the characterization and cDNA cloning of two alpha-amylase isozymes from larvae of the Western corn rootworm (Diabrotica virgifera virgifera LeConte). Larvae raised on artificial media have very low levels of amylase activity, and much higher levels are found in larvae raised on maize seedlings. At pH 5.7, the optimum pH for enzyme activity, the alpha-amylases are substantially but not completely inhibited by amylase inhibitors from the common bean (Phaseolus vulgaris) and from wheat (Triticum aestivum). Using the reverse transcriptase polymerase chain reaction (RT-PCR), we cloned two cDNAs with 83% amino acid identity that encode alpha-amylase-like polypeptides. Expression of one of the two cDNAs in insect cells with a baculovirus vector shows that this cDNA encodes an active amylase with a mobility that corresponds to that of one of the two isozymes present in larval extracts. The expressed enzyme is substantially inhibited by the same two inhibitors. We also show that expression in Arabidopsis of the cDNA that encodes the amylase inhibitor AI-1 of the common bean results in the accumulation of active inhibitor in the roots, and the results are discussed with reference to the possibility of using amylase inhibitors as a strategy to genetically engineer maize plants that are resistant to Western corn rootworm larvae.
Titarenko
E
E
Department of Biology, University of California San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0116, USA.
Chrispeels
M J
MJ
D000595:Amino Acid Sequence; D000818:Animals; D017360:Arabidopsis; D002460:Cell Line; D003001:Cloning, Molecular; D001517:Coleoptera / Q000201:enzymology*; D018076:DNA, Complementary; D007887:Fabaceae; D017344:Genes, Insect; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D007814:Larva / Q000201:enzymology; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000378:metabolism; D018517:Plant Roots / Q000378:metabolism; D010946:Plants, Medicinal; D017386:Sequence Homology, Amino Acid; D018411:Spodoptera / Q000166:cytology; D000516:alpha-Amylases / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism
D018076:DNA, Complementary; D007527:Isoenzymes; D010940:Plant Proteins; D000516:alpha-Amylases