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Submitted to: American Society of Plant Physiologists Meeting
Publication Type: Abstract Only Publication Acceptance Date: 5/18/1998 Publication Date: N/A Citation: N/A Interpretive Summary: Technical Abstract: Insects rely on a variety of midgut proteinases to catalyze the release of free amino acids from dietary protein and thereby provide nutrients essential for 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 erevealed 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. Proteinases isolated from dissected midguts of Hypera postica are being analyzed using gelatin-containing SDS-PAGE and class-specific PIs. A parallel approach is being pursued to clone cysteine proteinase (CP) genes, as previous studies have indicated that CPs play a prominent role in the digestive ability of this insect. DNA primer mixtures corresponding to evolutionarily conserved regions of amino acids within CPs were synthesized for use in PCR. Templates included DNA from H. postica, as well as DNA from the dipteran insects Tetanops myopaeformis (sugarbeet root maggot) and Drosophila melanogaster (fruit fly) as positive control. Fragments of about 500bp have been amplified from each of these templates. Subcloning followed by selection, propagation, and sequencing of individual clones derived from each template can be expected to reveal one or more cysteine proteinase genes for each organism. These genes will ultimately serve as tools to express recombinant CP for selecting potent inhibitors from a library of novel CP inhibitors. |
