Molecular cloning of plant secd cystatins and their antipest ettect

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This study purposes making basic contributions to breeding of edible seeds from a new aspect. For this, experiments were carried out to analyze a correlationship between the structure of seed cystatins and their activity for inhibiting cysteine proteinases, and also to verify their antipest functions, i.e., functions for regulating eating damages inflicted by insects such as Sitophilus zeamais. Corn, wheat, and soybean were used as materials. Cloning of cystatins from these seeds demonstrated the occurrence of cystatins highly homologous to oryzacystatin which had been found・ in rice seeds. Primary structures of these cystatins were 60-70% homologous to one another, each containing the amino acid sequence QXVXG as one of the active sites as seen in the case of any animal cystatins. However, the gene structures of these plant seed cystatins were greatly different from those of animal cystatins. This suggests that the plant cystatins should not be included in the three conventional families constructed based exclusively on animal cystatins, but in an independent "phytocystatin" family. For application purposes, cloned cystatin genes were introduced into Escherichia coli to produce cystatin proteins on a large scale. Enzymological assay showed that the cystatin proteins had potent papain-inhibitory activities. Interestingly, they also elicited potent activities to inhibit cysteine proteinases originating in the gut of the insects, Callosobruchus chinensis and Riptortus clavatus. The results provide strong evidence warranting the antipest effect of the seed cystatins.