用蛋白酶体亚单位直接抑制雌蚊繁殖势能的新探索

Mosquito-borne diseases cause serious mortality and morbidity in humans, and pose significant threats to public health. Recent years, there has been new outbreaks of mosquito-borne diseases (such as zika virus) around the world. Vector control is the primary method for controlling and preventing mosquito-borne diseases. The widely use of pesticide had resulted in the emergence and spread of resistance in mosquito populations. There is urgent needs for effective prevention and vector control strategies and tools. Only female mosquitoes are responsible for blood sucking and disease transmission. Research and develop new pesticide targeting female reproductive potential could be an efficient way for mosquito population suppression and mosquito-borne disease prevention.Our previous researches pointed out that proteasome subunit was only expressed at female abdomen and dsRNA based RNA interference targeting proteasome subunit resulted in reduced fertility in mosquitoes,suggesting that proteasome subunit play important roles in female reproduction. The central objectives of this new application are to examine the role of proteasome subunit in female mosquito reproduction and to evaluate the possibility of reaching population suppression by decreasing female fertility. The specific aims are: 1) to exam the function of proteasome subunitm in female reproduction using CRISPR-Cas9-mediated gene drive system, dsRNA interference system and pharmceutical inhibitors; and 2) to evaluated the efficacy of inhibiting female fertility on population suppression using semi-field experiment. We anticipated that the knowledge from this application will greatly facilitate the development of innovative strategies and tools for vector controlling management and help with vector-borne disease elimination.

蚊媒病一直是危害人类健康的重要公共卫生问题。近年来，全球不断有新的蚊媒病爆发（如寨卡病毒），蚊媒病的有效防控迫在眉睫。化学防制一直是媒介防制规划中的主要方法，然而蚊媒已对多种化学和生物杀虫剂产生抗性。因此研发可替代的新型杀虫剂, 尤其是寻找疾病传播媒介雌蚊繁殖势能调控的新方法，已成为目前媒介防制所面临的一大难题。申请人在上一个国家自然科学基金资助下，发现蛋白酶体催化亚单位（PSMB）在蚊特定阶段和部位表达，抑制蛋白酶体催化亚单位6表达能明显降低雌蚊的生殖力。本项目拟进一步研究PSMB在蚊生殖功能中的作用，采用CRISPR-Cas9系统构建PSMB敲除的转基因蚊，验证PSMB对蚊生殖功能的影响；并在半现场条件下观察PSMB抑制对靶标蚊的种群抑制效果。本研究的预期结果可望为研发基于直接控制雌蚊繁殖势能的新型无污染生物杀虫剂奠定基础，为蚊媒综合防制提供一种全新方法。

蚊媒病一直是危害人类健康的重要公共卫生问题。近年来，随着全球化、工业化和城市化的发展，蚊媒病的威胁在不断增长。且蚊媒杀虫剂抗性的产生，严重削弱了媒介化学防制的效果。从蚊媒生理出发，研究蚊媒生殖调控机制有望为研发新型杀虫剂提供有效靶点。本课题研究发现蛋白酶体B亚单位6（PSMB6）在雌蚊吸血后卵巢发育期、蚊胚胎发育期高表达。蛋白酶体特异性抑制剂MG-132处理雌蚊后，蚊产卵率、卵孵化率显著降低，且作用可持续至子2代。干涉雌蚊PSMB6的蛋白表达，雌蚊出现产卵时间延迟，产卵率、卵孵化率显著降低，卵筏中卵的个数显著减少，卵筏中卵排布稀疏，边缘不规整等现象。以上结果显示调控蛋白酶体亚单位PSMB6表达和活性对雌蚊卵巢和蚊卵有明显影响，提示PSMB6与雌蚊生殖密切相关。进一步机制研究显示，PSMB6可能通过弱化泛素介导的蛋白降解途径和脂质代谢通路来调节雌蚊生殖。为了进一步研究PSMB6在蚊生殖中的作用，我们用CRISPR/Cas9技术构建PSMB6敲除转基因蚊，虽然因技术原因未获得PSMB6敲除子代，但在PSMB6敲除母代蚊观察到雌蚊产卵量、幼虫数和孵化率均显著下降，且50%以上卵巢出现畸形，进一步证实PSMB6与雌蚊生殖密切相关。随后，我们改进了构建转基因蚊的操作技术。因CRISPR-Cas9 技术应用于非单细胞胚胎（如蚊卵）时，不能产生很好的生殖系转移能力，我们针对蚊生殖的特异性，即吸血后卵巢才开始发育，与宾夕法尼亚州立大学Rasgon教授实验室合作，使用基于受体介导 Cas9 卵巢特异性转运系统（即ReMOT Control系统）进行转基因蚊的构建，以提高Cas9蛋白进入卵细胞的效率，提高子代个体靶基因的敲除效率。目前转基因蚊的实验仍在进行中。本研究结果对于深入探讨雌蚊生殖机制，及建立基于雌蚊生殖力的蚊媒控制新策略提供了理论基础。