致倦库蚊中肠Cry48Aa/Cry49Aa新型双组份毒素功能性受体及其互作的分子基础

Lysinibacillus sphaericus biolarvicides based on the BinA/BinB toxin can easily induce target mosquito to develop high-level of resistance both in laboratory and under field conditions, and the frequent occurrence of high-level resistance to BinA/BinB toxin in mosquitoes has hindered the further application of L. sphaericus as microbial control agents. The Cry48Aa/Cry49Aa mosquitocidal two-component toxin was characterized from L. sphaericus strain and is uniquely composed of a three-domain Cry protein toxin (Cry48Aa) and a binary (Bin) toxin-like protein (Cry49Aa). However, the mode of action of Cry48Aa/Cry49Aa is still unknown, but a remarkable feature of this new two-component toxin is the capacity to overcome and delay Culex quinquefasciatus resistance to the Bin-based L. sphaericus biolarvicides. Therefore, these toxins have a surprising application potential as a new generation of biolarvicides used for the integrated control of mosquitoes. Our previous studies demonstrated that each Cry48Aa and Cry49Aa components carried the receptor binding site, respectively, which was different from the discovered binary toxin action mechanism such as BinA/BinB that single subunit carried receptor binding domain. So, Cry48Aa/Cry49Aa toxin may represent a new action mode of the binary toxin. In this study, Cry48Aa/Cry49Aa toxin’s functional receptors will be captured using Affinity Chromatography, Pull-down experiment, crosslink/toxin ligand transfer and LC/MS/MS methods. Then, the functional receptors isolated will be cloned and heterologously expressed, and their binding affinity with Cry48Aa/Cry49Aa protein is identified by Far-Western dot blot analysis and the binding kinetics in vitro. Next, physiological function of the toxin receptors will be studied using RNAi technology. The toxin’s Domain and Motif interaction with native receptors will be identified by molecular mutation technique and binding kinetics analysis, which is helpful to understand the molecular mechanism of the toxin-receptor interaction. This project will possibly elucidate a new action mode of the binary toxin, which will provide an insight into the understanding of the action of L. sphaericus mosquitocidal Cry48Aa/Cry49Aa toxin, and will help develop approaches for the resistance management and the continuable application of L. sphaericus-based larvicides in mosquito control in field.

长期大量使用以Bin毒素为基础的球形芽胞杆菌制剂已导致目标蚊虫产生高水平抗性，Cry48Aa/Cry49Aa新型双组份毒素能克服和延缓蚊虫对球形芽胞杆菌制剂的抗性进化，但目前对该毒素的作用机制不明。我们前期研究发现两毒素蛋白均携带有受体结合域，有别于Bin等二元毒素单个蛋白亚基携带受体结合域的作用方式，可能代表一种新的二元毒素作用模式。本项目通过亲和层析结合配体印迹等技术，从致倦库蚊幼虫中肠中捕获Cry48Aa和Cry49Aa结合蛋白，从结合特异性和亲和性等方面进行体外验证；利用RNAi技术研究毒素受体在蚊虫体内的生理功能；通过体外定点突变技术研究毒素-受体互作Domain及结合Motif，阐明毒素-受体互作的分子基础，以期揭示一种新的二元毒素作用模式。本项目可为阐明该毒素作用机制奠定理论基础，对制定抗性蚊虫治理策略及球形芽胞杆菌制剂在蚊虫控制方面的持续应用有重要意义。

长期大量使用以Bin毒素为基础的球形芽胞杆菌制剂已导致目标蚊虫产生高水平抗性，Cry48Aa/Cry49Aa新型双组份毒素能克服和延缓蚊虫对球形芽胞杆菌制剂的抗性进化，但目前对该毒素的作用机制不明。前期研究已发现两毒素蛋白均携带有受体结合域，有别于Bin等二元毒素单个蛋白亚基携带受体结合域的作用方式，可能代表一种新的二元毒素作用模式。本项目通过亲和层析结合配体印迹等技术，从致倦库蚊幼虫中肠中捕获一批Cry48Aa和Cry49Aa结合蛋白，利用LC-MS/MS方法分别对候选结合蛋白样品进行种类鉴定，发现氨肽酶（APN）、碱性磷酸酶（ALP）、α-淀粉酶（Amy）、α-葡萄糖苷酶（Glu）、肌动蛋白（Actin）、钙转运ATP酶（Cal）作为最优备选受体蛋白，利用RNA干扰技术对其蛋白表达产物的进行沉默并结合生物测定结果，确定ALP63、APN49和Glu71可能是Cry48Aa或Cry49Aa毒素的受体蛋白，并对其进行原核表达和体外结合动力学研究，发现Cry48Aa毒素与Glu 71蛋白能够形成典型的饱和曲线，有较高的特异性结合能力，其最大结合量（Bmax）为42.6±4.94 pmol/mg，解离常数（Kd）为48.66±15.66 nmol。而Cry49Aa毒素呈现非特异性结合，最终确定Glu71蛋白是Cry48Aa毒素的受体。通过体外定点突变技术研究毒素-受体互作Domain及结合Motif，发现Cry48Aa毒素的N-端结构域负责和Cry49Aa毒素互作并决定杀蚊毒力，Cry49Aa毒素的N-端Domain负责和Cry48Aa毒素互作，C端Domain负责和蚊中肠受体结合，其中C91位Cys残基突变影响毒素的产量，C183突变影响毒素与Cry48Aa的结合，同时也降低毒素的杀蚊活性及与中肠受体蛋白的结合活性。本研究结果可为阐明该毒素作用机制奠定理论基础，对制定抗性蚊虫治理策略及球形芽胞杆菌制剂在蚊虫控制方面的持续应用有重要意义。