蛋白磷酸酶DSP3调控Bt抗性小菜蛾MAPK信号途径激活的内稳态机制研究

Bioinsecticides and transgenic crops based on Bacillus thuringiensis (Bt) are crucial for pest control in the field. However, insufficient understanding of the molecular mechanisms of insect resistance to Bt seriously jeopardizes the long-term sustainable utilization of Bt products. Our previous studies have found that Bt resistance in Plutella xylostella (L.) was caused by the activation of MAPK signaling pathway via kinase phosphorylation. Moreover, the homeostasis of MAPK signaling pathway is controlled by both phosphorylation of protein kinases and dephosphorylation of protein phosphatases. Therefore, on this basis, the current foundation aims to further carry out the study on homeostatic mechanism underlying activation of MAPK signaling pathway mediated by dephosphorylation of protein phosphatases. Firstly, the protein phosphatase DSP3, which is differentially expressed at phosphorylation levels between Bt susceptible and resistant strains identified by phosphoproteomics, will be cloned and quantified among different strains. Subsequently, we will use RNAi and CRISPR/Cas9 to verify the pivotal role of DSP3 in regulating the homeostasis of MAPK signaling pathway in Bt-resistant P. xylostella, which will contribute to elucidating the homeostatic mechanism of MAPK signaling pathway and to further revealing the MAPK-mediated molecular mechanism of Bt resistance in insects. The results of this study will be of great theoretical and practical significance for management of field-evolved Bt resistance and for development and sustainable application of Bt products.

基于苏云金芽胞杆菌Bt开发的生物源杀虫剂和转基因作物对于田间害虫防治至关重要，而对害虫Bt抗性分子机制认识的不足严重制约着Bt产品的可持续利用。申请者前期研究发现小菜蛾Bt抗性是由MAPK信号途径蛋白激酶磷酸化激活导致，而MAPK信号途径激活的内稳态主要是由蛋白激酶磷酸化和蛋白磷酸酶去磷酸化共同控制。因此，本项目拟在此基础上继续开展蛋白磷酸酶去磷酸化调控其内稳态机制的研究。首先对磷酸化蛋白质组鉴定出的差异蛋白磷酸酶DSP3在Bt敏感和抗性小菜蛾种群中进行基因克隆和定量分析；随后使用RNAi和CRISPR/Cas9技术验证DSP3在调控Bt抗性小菜蛾MAPK信号途径内稳态机制中的关键作用，从而阐明Bt抗性小菜蛾MAPK信号途径激活的内稳态机制，进一步揭示该信号途径介导的昆虫Bt抗性分子机制。研究结果对于田间害虫Bt抗性治理以及Bt产品的开发与可持续应用具有重要的理论和实践意义。

基于苏云金芽胞杆菌(Bt)产生的杀虫蛋白开发的微生物杀虫剂和转基因作物在害虫田间防治中发挥举足轻重的作用，然而，害虫不断对Bt生物产品进化出抗性。对害虫Bt抗性分子机制认识的不足制约着Bt生物产品的可持续应用。小菜蛾是在世界范围分布的十字花科作物主要害虫之一，同时，它还是第一个被报道在田间对Bt微生物杀虫剂产生抗性的害虫。由于其基因组的充分解析，小菜蛾成为研究Bt抗性分子机制的生物模型。申请者前期研究发现MAPK信号途径激活进而反式调控多个抗性相关基因导致小菜蛾对Bt Cry1Ac产生高抗性。然而，对于MAPK信号途径是如何磷酸化激活的以及这种磷酸化水平的稳态是如何维持的关键科学问题尚未阐明。在此基础上，本项目解析了小菜蛾中MAPK信号途径的激酶成员组成，阐明了MAPK信号途径在不同节肢动物中的保守性，明确了Bt Cry1Ac抗性小菜蛾中调控下游抗性相关基因的MAPK信号途径磷酸化拓扑网络，揭示了MAPK信号途径磷酸化抑制剂可作为Bt抗性治理的重要策略，阐明了磷酸酶DSP3可维持关键MAPK信号途径磷酸化内稳态的分子机制，构建了小菜蛾Bt Cry1Ac抗性分子机制的组学调控网络图，进一步阐明了小菜蛾Bt Cry1Ac抗性分子机制。研究结果对于充分解析害虫Bt抗性分子机制，田间农业害虫Bt抗性的监测和治理以及新型Bt生物产品的研发和可持续应用具有重要的理论和实践意义。