Expression of the soybean (Kunitz) trypsin inhibitor in transgenic tobacco: effects on larval development of Spodoptera litura

The coding region of the Ti^sub a^ allelic form of the soybean (Kunitz) trypsin inhibitor gene has been introduced, as a transcriptional fusion with the CAMV 35S promoter, into tobacco. Southern analysis of DNA extracted from progeny (Fl) plants confirmed that an intact copy (or copies) of the gene is integrated into the tobacco genome. Gel filtration column chromatography has been used to partially purify the inhibitor from leaves of transgenic tobacco and inhibition assays revealed that the protein can inhibit both bovine trypsin, and trypsin-like (BApNA-hydrolysing) activity extracted from Spodoptera litura digestive tracts. SDS-PAGE and western blotting determined that the inhibitor accumulates as a protein of ca. 20kD in transgenic leaf tissue. The protein has been purified to homogeneity using reverse-phase column chromatography, and subsequent N-terminal sequencing revealed that the inhibitor is processed in tobacco leaf tissue by the removal of the N-terminal leader sequence. Insect feeding trials, using neonate larvae of S. litura, have been conducted with leaf tissue excised from transgenic progeny plants that either accumulated the inhibitor or from control (non-transgenic) plants. These trials established that, when compared with insects fed non-transformed leaf tissue, larvae fed transgenic leaf tissue demonstrated significantly greater mortality, and the survivors grew more slowly in terms of weight gain over time. These results are interpreted with respect to current opinion on the use of proteinase inhibitors as insect pest resistance factors in transgenic plants.[PUBLICATION ABSTRACT]