Analysis of the mode of action of fungicides and drug design based on structural analysis of pathogenicity related genes products from plant pathogenic fungi

基于植物病原真菌致病相关基因产物结构分析的杀菌剂作用方式及药物设计

• 批准号: 07556080
• 负责人: FURUSAWA Iwao
• 金额: $ 3.14万
• 依托单位: KYOTO UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (A)
• 财政年份: 1995
• 资助国家: 日本
• 起止时间: 1995 至 1997
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07556080/
• 关键词: Melanin; Carpropamid; Scytalone; Colletotrichum lagenarium; Magnaporthe grisea; Trihydroxynaphthalene; Appressoria; Magnaporthe grisea; トリヒドロオキシナフタレン; メラニン合成酵素; サイタロン脱水酵素遺伝子; p-ジフェノールオキシダーゼ; 酵素精製; 結晶解析

Melanin biosynthesis is essential for appressorial penetration of Colletotrichum lagenarium. Three major melanin biosynthetic enzymes, polyketide synthase PKS1, scytalone dehydratase SCD1 and 1,3,8-trihydroxynaphthalene reductase THR1 were expressed in E.coli and purified. We used either pMAL and pET system for the heterologous expression of proteins and purified by affinity chromatography. And in the case of PKS1, we constructed an expression vector for Aspergillus oryzae and identified the products by PKS1. We analyzed the mode of action of an novel anti-rice blast fungicide carpropamid by using purified enzymes. First, we established in vitro reaction system and then analyzed the inhibitory activity against the dehydration reaction by carpropamid. The result obtained was that carpropamid directly act to the enzyme and thus bringing about inhibition of melanin biosynthesis. Further we analyzed multimer formation of SCD1 by yeast two-hybrid system. The result obtained indicated that SCD1 enzyme associates to each other and would form multimer. Lastly we prepared polyclonal antibody against purified PKS1, SCD1 and THR1 and immuno-blot analysis of those melanin biosynthetic enzymes indicated that there would be a kind of post-translational regulation for the enzyme activity.

黑色素生物合成对于炭疽病菌的附着渗透至关重要。在大肠杆菌中表达并纯化了三种主要的黑色素生物合成酶：聚酮合酶PKS1、scytalone脱水酶SCD1和1,3,8-三羟基萘还原酶THR1。我们使用 pMAL 和 pET 系统进行蛋白质的异源表达，并通过亲和层析进行纯化。以PKS1为例，我们构建了米曲霉的表达载体，并通过PKS1鉴定了产物。我们使用纯化的酶分析了新型抗稻瘟病杀菌剂卡磺酰胺的作用方式。首先，我们建立了体外反应体系，然后分析了卡磺酰胺对脱水反应的抑制活性。得到的结果是卡磺酰胺直接作用于该酶，从而抑制黑色素生物合成。进一步我们分析了酵母双杂交系统 SCD1 多聚体的形成。所得结果表明SCD1酶相互缔合并形成多聚体。最后我们制备了针对纯化的PKS1、SCD1和THR1的多克隆抗体，并对这些黑色素生物合成酶进行了免疫印迹分析，表明酶活性存在一种翻译后调节。

项目成果

Gento Tsuji: "Carpropamid,an anti-rice blast fungicide,inhibits scytalone dehydratase activity and appressorial penetration in Colletotrichum lagenarium" Pesticide Biochemistry and Physiology. 57. 211-219 (1997)

Gento Tsuji：“Carpropamid，一种抗稻瘟病杀菌剂，可抑制炭疽病菌中的双镰孢酮脱水酶活性和附着渗透”农药生物化学和生理学。

Gento Tsuji: "Carpropamid,an anti-rice blost fungicide,inhibits scytalone dehydratase activity and appressorial penetration in colletofrichum lagenarium" Pesticide Biochemistry and Physiology. 57. 211-219 (1997)

Gento Tsuji：“Carpropamid，一种抗稻瘟病杀菌剂，抑制镰刀菌酮脱水酶活性和粘连渗透”农药生物化学和生理学。

Tsuji, G., T.Takeda, I.Furusawa, O.Horino and Y.Kubo: "Carpropamid, an anti-rice blast fungicide, inhibits scytalone dehydratase activity and appressorial penetration in Colletotrichum Lagenarium." Pesticide Biochemistry and Physiology. 57. 211-219 (1997)

Tsuji, G.、T.Takeda、I.Furusawa、O.Horino 和 Y.Kubo：“Carpropamid 是一种抗稻瘟病杀菌剂，可抑制 Colletotrichum Lagenarium 中的 scytalone 脱水酶活性和附着渗透。”