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

黑素的生物合成对炭疽菌的附着渗透至关重要。三种主要的黑色素生物合成酶：聚酮合成酶PKS1、胞糖酮脱水酶SCD1和1,3,8-三羟基萘还原酶THR1在大肠杆菌中表达并纯化。我们分别用pMAL和pET系统进行蛋白的异源表达，并通过亲和层析纯化。以PKS1为例，构建了米曲霉表达载体，并利用PKS1对产物进行了鉴定。用纯化酶分析了新型抗稻瘟病杀菌剂丙烯酰胺的作用方式。首先建立了体外反应体系，分析了丙烯酰胺对脱水反应的抑制活性。结果表明，卡丙胺直接作用于该酶，从而抑制黑色素的生物合成。进一步分析了酵母双杂交系统中SCD1的多次形成。结果表明，SCD1酶相互结合，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免疫印迹分析，结果表明这些酶的活性可能存在翻译后调控。

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.