基于祖先序列重建的Bt Cry2A蛋白杀虫功能进化分子机制研究

获得新型、高效、广谱且与现已商业化应用的杀虫蛋白无交互抗性的Bt杀虫蛋白是我国农业害虫防控的重大需求。目前，通过蛋白质工程对现有杀虫蛋白进行改造是Bt领域的研究热点。当代蛋白质是祖先蛋白质谱系随时间不断累积突变进化而来，基于祖先序列重建的方法重现蛋白质功能的历史突变轨迹，能够快速找到功能演化的决定因素，为蛋白改造提供新途径。申请人前期研究发现Bt杀虫晶体蛋白的祖先蛋白对靶标害虫杀虫活性更高，并且出现了功能分割的现象。本项目在前期工作的基础上，通过祖先蛋白和当代蛋白杀虫活性和杀虫特异性的比较分析，结合分子进化特征，重建杀虫蛋白功能演化轨迹；基于定点突变等方法解析其功能进化的分子机制并进行蛋白改造，以期获得活性提高或杀虫谱扩大的新型Bt杀虫蛋白。本项目的实施将有望揭示Bt杀虫晶体蛋白功能进化的分子机制，同时将丰富我国抗虫基因资源，为今后Bt杀虫蛋白改造开辟新途径。

Obtaining novel insecticidal proteins of Bacillus thuringiensis with high activity, broad insecticidal spectrum or no cross resistance to commercialized proteins is a major demand of agricultural insect pest control in China. Currently, the modification of existing insecticidal proteins based on protein engineering is a hotspot on Bt. Protein function evolved as mutations accumulated through time in ancestral protein lineages. Recapitulating mutational trajectories that occurred during evolution based on the ancestral sequence reconstruction method could quickly identify the determinants of protein function evolution and provide new ideas for protein modification. Our previous study found that Bt ancestor insecticidal protein showed higher insecticidal activity against target insect pests compared with modern insecticidal proteins, and appeared the feature of insecticidal function partitioning. Based on the previous work, in this project, the functional evolution process of Bt insecticidal protein will be reconstructed through comparing the insecticidal activity and specificity of ancestral proteins and modern proteins, combined with molecular evolution characteristics. Furthermore, the molecular mechanism of insecticidal functional evolution will be clarified and novel insecticidal proteins with higher activity and broader insecticidal spectrum will be obtained through site-directed mutation and other molecular designing methods. This project will reveal the molecular mechanism of Bt insecticidal protein function evolution, enrich China’s insect-resistant gene resources and open up new ways for future Bt insecticidal protein modification.