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

黑色素的生物合成是炭疽菌附着体穿透所必需的。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，即聚酮合成酶PKS1、半胱氨酸脱水酶SCD1和1，3，8-三羟基萘还原酶THR1。我们使用pMAL和pET系统进行异源表达，并用亲和层析进行纯化。以PKS1为例，构建了米曲霉的表达载体，并对表达产物进行了鉴定。利用纯化的酶制剂对新型抗稻瘟病杀菌剂丙丙胺的作用机理进行了分析。首先，我们建立了体外反应体系，并分析了其对丙丙胺脱水反应的抑制活性。得到的结果是，卡丙胺直接作用于该酶，从而抑制黑色素的生物合成。此外，我们还利用酵母双杂交系统分析了SCD1多聚体的形成。结果表明，SCD1酶之间相互作用，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，对这些黑色素生物合成酶的免疫印迹分析表明，这些酶的活性可能存在一种翻译后调节。

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.