昆虫TopoI氨基酸多型性及天然非喜树碱类TopoI抑制剂研究

It is important to find the potential specific target and lead compounds from plants. Our previous research works indicated that DNA topoisomerase I （Topo I） from Spodoptera exigua Hübner is the target of camptothecin (CPT) and its derivates, which suggested a potential specific target for the control of insects. Researching systemically the function of Topo I and the structure-activity relationships between Topo I and its inhibitors will benefit the development and application of this specific target and its inhibitors. Based on our previous research works, we will focus on studying the effect of amino acid polymorphisms of Topo I on its structure and function, finding the sensitive polymorphic amino acid sites to CPT. According to these results, the sensitivity of the pests to CPT and its derivatives will be evaluated through cloning the genetic fragments including amino acid polymorphisms of TopoI from some important insects. Therefore, the controlling spectrum of CPT and its derivatives can be illustrated, which will provide important informations for the rational use of CPT and its derivatives, and for developing more environment-friendly insecticides based on CPT. Meanwhile, the compounds from animals, microorganisms and plant will be screened based on amino acid polymorphisms of Topo I to discovery more Topo I inhibitors, which can be used to control crop pests or to provide more potential lead compounds.

从植物中发现特异性作用靶标和先导化合物，对创制具有自主知识产权的环境友好型农药意义重大。本课题组研究证明甜菜夜蛾拓扑异构酶I （Topo I）是喜树碱类化合物的作用靶标，系统性地开展该靶标与喜树碱类抑制剂的关系研究将有利于此特异性靶标农药的研发与应用。本项目将在前期工作基础上，通过对昆虫Topo I氨基酸多型性及其对Topo I结构和功能的影响研究，确定昆虫Topo I对喜树碱类抑制剂敏感的氨基酸位点；克隆我国主要农业害虫Topo I氨基酸多型性基因片段，测定和评价其对喜树碱类抑制剂的敏感性，明确喜树碱及衍生物的防治害虫谱，促进以喜树碱为先导化合物的环境友好型特异性靶标新农药的开发；同时，利用昆虫Topo I氨基酸多型性与Topo I抑制剂的互作机制，对来自动物、植物微生物的天然化合物进行活性筛选，发现可用于防治农业害虫的Topo I抑制剂，为新农药的创制贮备更多具有发展潜力的先导化合物。

拓扑异构酶 I（topoisomerase I， Topo I）是生物体内参与 DNA 复制，转录，修复和重组等关键过程的酶，目前被认为是喜树碱类化合物唯一的作用靶标，其结构与喜树碱类抑制剂的敏感性密切相关。在昆虫中，Topo I 基因在与喜树碱相互作用直接或间接相关的氨基酸位点（V420， L530， A653 和 T729）上存在多型性，而这些多型性对其喜树碱类化合物敏感性的影响及其机理却不清楚。本项目通过定点突变、蛋白表达纯化与活性测定揭示甜菜夜蛾Topo I在530，653和729位的氨基酸多型性通过改变Topo I对底物双链DNA的解旋速率，而影响其对喜树碱及其衍生物敏感性的机理；通过喜树碱及其衍生物抑制活性测定，证明具有L530P和/或A653T型Topo I的昆虫对喜树碱及其衍生物敏感性降低，而S729T则提高了昆虫对喜树碱及其衍生物的敏感性；通过对昆虫Topo I基因比较分析揭示其对喜树碱类衍生物的敏感性进化；通过建立以野生型和突变型Topo I为平台的筛选体系，对11类非喜树碱类抑制剂进行活性筛选，发现其均无高活性，间接证明昆虫Topo I作为靶标的特异性。这些研究结果对指导喜树碱及其衍生物在农业害虫防治中的科学合理使用和以喜树碱为先导化合物的环境友好型特异性新农药的开发具有重要的理论意义；同时对探索把 Topo I 作为害虫防治的特异性生态安全性靶标的潜力与可行性具有重要的指导意义。