ISOLATION OF A CYSTEINE PROTEINASE CDNA FROM THE ALFALFA WEEVIL AND ANALYSIS OF ITS MIDGUT PROTEINASES

Authors: Wilhite, Stephen; Smigocki, Anna; Elden, Thomas

Submitted to: Mid Atlantic Plant and Molecular Biology Proceedings
Publication Type: Abstract Only
Publication Acceptance Date: 7/9/1998
Publication Date: N/A
Citation: N/A

Interpretive Summary:

Technical Abstract: Insects rely on a variety of midgut proteinases to break-down dietary protein and thereby provide nutrients essential for their normal growth and development. A potential for insect control has been demonstrated in laboratory studies involving the expression of proteinase inhibitor (PI) genes in transgenic plants. However, insects have revealed an ability to compensate for lost proteinolytic activity by enhancing production of proteinases insensitive to the introduced PI. Thus, there is a need to characterize the individual proteolytic enzymes within an insect in order to pursue a directed control strategy in which each proteolytic activity is specifically targeted for inhibition. We are conducting both biochemical and molecular cloning experiments to elucidate the digestive proteinases of Hypera postica. Gelatin-containing SDS-PAGE of weevil midgut extracts has revealed one major and several minor size-classes of proteolytic activity. The large majority (70-80%) of proteolytic activity appears to result from cysteine proteinases in the midgut extract, as revealed by inhibition of the enzymatic activity with class-specific protease inhibitors. Of interest from the standpoint of pest control, the recombinant rice inhibitors OCI and OCII were similarly effective at inhibiting proteolytic activity as the potent, irreversible cysteine proteinase inhibitor E-64. One cysteine proteinase clone has been identified in a random sampling of 10 lambda clones from an H. postica midgut-specific cDNA library. DNA sequencing of the insert has revealed a full-length cDNA (hcp1) encoding a predicted protein (HCP1) of 324 amino acids. This putative digestive enzyme is highly similar to cathepsin L-type cysteine proteases, and is predicted to play a major role in the assimilation of dietary protein in the alfalfa weevil.