基于抑制几丁质合成的新型杀虫剂靶标的筛选与鉴定

The wide use of insecticide has resulted in the emergence and spread of resistance in mosquito populations, which become a major obstacle in mosquito vector control programs. There is urgent needs for mosquito control strategies and tools. As chitin plays an important role in the process of growth and development of mosquito, inhibition of the chitin biosynthesis has attracted much research interest because of its great potential in the comprehensive management of mosquito control. Our previous researches pointed out that RNA interference (RNAi) of Tre-1 resulted in increased mortality and decreased emergence rate. Moreover, the chitin content of emerged dsRNA adults was decreased compared with the control. In this project, the functional characteristics of all eight enzymes involved in the chitin biosynthesis pathway will be analyzed comprehensively. Moreover, the functions and potential mechanisms of the key enzyme involved in mosquito growth and development will be further explored by CRISPR-Cas9-mediated gene drive system. We anticipated that the knowledge from this application will provide insight into the biological functions of enzymes involved in the chitin biosynthesis pathway and greatly facilitate the development of innovative strategies and tools for mosquito controlling management.

化学杀虫剂连续大量使用导致抗药性的发生和发展，已成为蚊媒防治的巨大障碍。研发可替代的新型杀虫剂是目前蚊媒防治的一大难题。几丁质在蚊生长发育过程中发挥着重要作用，且不存在于高等动物中，因此其合成途径是新型杀虫剂设计的安全靶标。然而几丁质合成途径的分子机理还有待深入研究。申请人在上一个国家自然科学基金资助下获得几丁质合成通路起始酶Tre-1。 进一步功能研究发现抑制Tre-1的表达，蚊死亡率增高，羽化率降低，羽化后成蚊几丁质含量减少，提示Tre-1在蚊生长发育中发挥重要作用。本项目拟进一步对几丁质合成通路中的8个酶进行系统和深入研究，筛选出对蚊生长发育阶段影响最大的关键酶；并构建CRISPR-Cas9关键酶敲除蚊，深入探讨其作用及可能机制。本项目预期结果有助拓宽对蚊几丁质合成通路相关酶生物学功能的认识，为研制新型无污染杀虫剂奠定基础，并为探索控制蚊虫新方法提供科学依据。

化学杀虫剂连续大量使用导致抗药性的发生和发展，已成为蚊媒防治的巨大障碍。深入探讨抗药性分子机制，研发可替代的新型杀虫剂是目前蚊媒防治的一大难题。本项目从几丁质合成通路筛选出对蚊生长发育影响最大的关键酶CHSA，发现在淡色库蚊不同发育阶段对该基因进行干涉可造成不同的损伤表型：幼虫阶段干涉CHSA导致大量蚊出现蜕皮、化蛹和羽化失败等致死性表型；蛹期干涉CHSA后，蚊在蛹到成蚊羽化阶段几乎全部死亡。几丁质染色实验发现CHSA干涉组新几丁质层显著变薄，且旧几丁质层与上皮细胞无法分离。透射电镜发现干涉组新表皮生成延迟，且新旧表皮分离困难；在成蚊阶段干涉CHSA未发现明显的畸形和死亡，但表皮存在变薄现象。上述结果表明在幼虫及蛹阶段干涉CHSA对蚊生长发育有明显的抑制效果，提示该基因在蚊生活史不同发育时期转换阶段发挥着重要作用，值得我们进一步研究其在现场实际应用。本项目首次对CHSA的功能和机制进行了较为全面的探讨，为建立更有针对性的安全杀虫剂提供新的理论依据，具有潜在的应用价值。