Recombinant soybean cysteine proteinase inhibitors: Chemical characteristics and effectiveness against select crop pests

Lalitha Subramanian, Purdue University

Abstract

The overall goal of this research was to partially characterize three recombinant soybean cysteine proteinase inhibitors (rSCPIs) L1, R1 and N2, and to assess their potential to inhibit the growth and development of three major agricultural crop pests known to utilize cysteine proteinases (CPs) for their digestion: Western corn rootworm (WCR), Colorado potato beetle (CPB) and cowpea weevil (CW). Partial characterization of the three 10,000 dalton rSCPIs indicated that they were heat labile, had activity over a broad pH range, and were easily digestible by three human digestive enzymes, trypsin, chymotrypsin and pepsin. In vitro experiments showed that CP activities in the crude gut extracts of the WCR, CPB, and CW were inhibited to various degrees by the three rSCPIs. Of the three rSCPIs tested, N2 was most effective in inhibiting the crude gut extract of WCR (90% inhibition at 10−5M; 50% inhibition at 5 × 10−8 M), CPB (90% inhibition at 10−5M; 50% inhibition at 5 × 10−8M), and CW (90% inhibition at 10 −4M; 50% inhibition at 3 × 10−7M). The L1 was the least potent of the three CPIs tested, with 50% inhibition at 5 × 10−6M of the crude gut extracts of WCR. Results of in vivo studies testing the effects of the three rSCPIs on the vital growth parameters of WCR, CPB, and CW concurred with the findings of in vitro experiments. Studies done on the effect of rSCPIs in the native milieu of plants cells (potato leaves and corn roots) raised certain concerns due to observation of apparent interaction between the rSCPIs and the plant proteases. All three rSCPIs were prone to enzymatic degradation when exposed to the crude luminal contents of WCR, CPB and CW, showing increased protein breakdown and loss of inhibition activities with increased exposure time. The same result was reflected as a decrease in the inhibitory activity of the three rSCPIs monitored over the same exposure time intervals assessed with the 3H-metHb. Although with some reservations, results in summary indicate that rSCPIs are good candidates for conferring insect resistance in plants.

Degree

Ph.D.

Advisors

Nielsen, Purdue University.

Subject Area

Food science|Agronomy

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