Analysis of Insect Gut Enzymes and Verification of Regulatory Effects of Specific Inhibitors against Eating Damages on Food Seeds.

昆虫肠道酶分析和特定抑制剂对食品种子食害的调节作用验证。

• 批准号: 10556078
• 负责人: ABE Keiko
• 金额: $ 4.99万
• 依托单位: The University of Tokyo
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1998
• 资助国家: 日本
• 起止时间: 1998 至 1999
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10556078/
• 关键词: Coleoptera; insect gut proteinase; cysteine proteinases; Sitophilus cysteine proteinase; Oryzacystatin; 立体構造

Detailed analysis were conducted of cysteine proteinases of Sitophilus zeamais (Coleoptera), a rice-seed insect pest, to elucidate their properties and physiological functions at the molecular level. It was found that the cysteine proteinases comprise multiple molecular species originating from a gene family and also that one of the species is a central enzyme. involved in damaging the seeds. The result made us try to develop a transgenic rice cultivar with oryzacystatin or corn cystatin expressed at a higher level. For the trial, we first fused a cystatin cDNA and a hyglomycin-resistant gene to Ca MV35S promoter and then introduced the fusion gene to rice protoplasts by electroporation or using an agrobacterium-mediated system. The consequently regenerated transformant was found to express 10-15 times more cystatin than the non-transformant (normal rice) used as control. The cystatin expressed in the transformant seeds, when purified, showed a strong inhibitory activity (ICィイD250ィエD2 : 〜10ィイD1-8ィエD1M) toward cathepsin H as well as toward the Sitphilus cysteine proteinase. We then produced transgenic rice of this type at a field scale and harvested the seeds. An investigation to find out the antipest and even lethal effects of the seeds themselves to successfully progressing.

对水稻种子害虫 Sitophilus zeamais（鞘翅目）的半胱氨酸蛋白酶进行了详细分析，以在分子水平上阐明其特性和生理功能。发现半胱氨酸蛋白酶包含源自基因家族的多个分子种类，并且其中一个种类是中心酶。参与破坏种子。这一结果促使我们尝试培育高表达谷胱抑素或玉米胱抑素的转基因水稻品种。在该试验中，我们首先将半胱氨酸蛋白酶抑制剂 cDNA 和潮霉素抗性基因融合到 Ca MV35S 启动子上，然后通过电穿孔或使用农杆菌介导的系统将融合基因引入水稻原生质体。结果发现，再生的转化体表达的半胱氨酸蛋白酶抑制剂比用作对照的非转化体（正常水稻）多10-15倍。转化体种子中表达的半胱氨酸蛋白酶抑制剂在纯化后，对组织蛋白酶 H 以及 Sitphilus 半胱氨酸蛋白酶表现出强抑制活性（ICィイD250ィエD2：〜10ィイD1-8ィエD1M）。然后，我们在田间规模生产了这种类型的转基因水稻并收获了种子。一项旨在找出种子本身对成功进展的抗虫甚至致命影响的调查。

项目成果

N. Kudo: "Crystallization and preliminary X-ray diffraction studies of a rice cysteine proteinase inhibitor, oryzacystatin-I"J. Biochem.. 123. 568-570 (1998)

N. Kudo：“水稻半胱氨酸蛋白酶抑制剂 oryzacystatin-I 的结晶和初步 X 射线衍射研究”J。

M. Asano-Miyoshi: "Identification of taste tissue-specific cDNA clones from a subtraction cDNA library of rat circumvallate and foliate papillae"J. Biochem.. 124. 927-933 (1998)

M. Asano-Miyoshi：“从大鼠外周和叶状乳头的消减 cDNA 文库中鉴定味觉组织特异性 cDNA 克隆”J.

M.Asano-Miyoshi: "Identification of taste tissue-specific cDNA clones from a subraction cDNA library of rat circumvallate and foliate papillae"J.Biochem.. 124. 927-933 (1998)

M.Asano-Miyoshi：“从大鼠周乳头和叶状乳头的亚分 cDNA 文库中鉴定味觉组织特异性 cDNA 克隆”J.Biochem.. 124. 927-933 (1998)

J.Funaki,: "Purification and characterization of a neutral protease that contributes to the unique flavor and texture of tofu-misozuke." J.Food Biochem.21. 191-202 (1997)

J.Funaki：“中性蛋白酶的纯化和表征，有助于形成豆腐味噌的独特风味和质地。”

T. Misaka: "A gustatory cyclic nucleotide-gated channel. CNGgust is expressed in the retina"NeuroReport. 10. 743-746 (1999)

T. Misaka：“味觉环核苷酸门控通道。CNGgust 在视网膜中表达”NeuroReport。