Molecular cloning of plant secd cystatins and their antipest ettect

这项研究的目的将作出基本贡献，以从一个新的方面培育出可再生种子。由于这个原因，实验已经进行了分析，以分析种子细胞的结构及其抑制性半胱氨酸蛋白质的活动之间的相关关系，并核实其抗功能，一. E，用于调节食用损害的函数，如Sitophilus zeamais。玉米、小麦和大豆都被用作材料。从这些种子中克隆出细胞抑素，演示了细胞抑素的出现高度同源性到哪一种已经找到的oryzacystatin·在大米种子中。这些细胞质的主要结构与另一个细胞质相似60- 70%相同，每个细胞质含有氨基酸序列QXVXG，即在任何动物细胞质的情况下都可以看到的一个。However，这些植物种子的半胱氨酸的基因结构与那些动物半胱氨酸有很大的不同。This suggests that the plant cystatin should not be included in the three conventional fManagement constructed based exclusively on animal cystatin， but in an independent“phytocystatin”family。为了应用目的，克隆的半胱氨酸基因被引入到大肠杆菌中，并在大尺度上生产半胱氨酸蛋白质。Enzymological assay显示了cystatin蛋白质具有潜力的纸张-抑制剂活性。有趣的是，它们也很有可能在昆虫的肠道、中国卡洛索布鲁丘斯和里普托图斯克拉瓦图斯中抑制蛋白质的活性。结果提供了强有力的证据警告种子细胞的抗效应。

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.