对鳞翅目害虫高效的Bt Cry1和Cry2类杀虫蛋白的定向改造

Bacillus thuringiensis (Bt) pesticides and genetically modified crops expressing Bt insecticidal genes play an important role in insect pests control. For efficient and sustainable control of important agricultural pests, the new insecticidal proteins with activity against the new target pests, high toxicity or no cross resistance to commercialized toxin are needed. In recent years, with the growing number of novel Bt insecticidal genes found, it is much more difficult to discover novel Bt insecticidal proteins from wild Bt isolates, while remolding known proteins by molecular designing is an optional pathway. .Based on the determining the insecticidal activity of Cry1 and Cry2 type proteins, we will rationally remold the proteins by site-direct mutagenesis through analyzing the charge and other characteristics of key amino acids. Through in vitro and in vivo interaction analysis of interaction between Cry proteins and receptors, we will reveal “the relationship between structure and function”, optimize the theory of “insecticidal protein-receptor interaction”, and propose a model about site-direct remolding of Cry1 and Cry2 proteins. The result of this program not only can comprehend "Bt- insect" cooperative coevolution in molecular level, but also provides new gene resources of Bt pesticides and genetically modified crops in China.

目前，Bt杀虫剂与转Bt杀虫基因作物在害虫防治中发挥了重要作用。为了对农业重大害虫进行高效持续防治，就需要获得具有新杀虫谱、高活性或者与已商业化应用的杀虫蛋白无交互抗性的新型Bt杀虫蛋白（Cry蛋白）。近年来随着新Bt基因挖掘数量不断增长，从自然菌株中发现新基因的难度越来越大，而通过分子设计定向改造现有基因是一条有效途径。.本项目在明确Cry1和Cry2类备选蛋白杀虫活性的基础上，通过分析Cry蛋白杀虫关键位点的氨基酸带电性等特性，结合氨基酸突变等技术方法定向改造蛋白。然后通过Cry杀虫蛋白与受体蛋白的体外相互作用分析以及在昆虫体内验证等手段，揭示Cry蛋白与昆虫受体的“构效关系”，完善“杀虫蛋白-受体”互作理论，提出Cry1和Cry2类蛋白定向改造的理论模型。本项研究将有助于在分子水平解析“Bt-昆虫”的协同进化，而且将为我国Bt杀虫剂、转基因抗虫植物研发提供新的基因资源。

苏云金芽胞杆菌（Bacillus thuringiensis，Bt）是全世界应用最广的杀虫微生物，其主要杀虫物质晶体蛋白基因广泛应用于抗虫作物中可以有效防控多种农业害虫，但杀虫活性不足限制了很多杀虫基因的应用。因此本项目通过蛋白结构理性设计，对Cry杀虫蛋白的结构域II和III区域的氨基酸进行了定向改造，获得了一批对草地贪夜蛾和棉铃虫等鳞翅目害虫高活性的改造蛋白。然后通过ZDOCK对Cry蛋白的关键位点与潜在受体蛋白进行分子对接，初步总结了Bt杀虫蛋白与靶标害虫中肠受体的构效关系，为后续其它杀虫蛋白的定向改造奠定了坚实的基础。.本项目完成期间，在昆虫学顶尖刊物Pest Management Science等国内外学术期刊发表论文8篇，4项国家发明专利获得授权。项目相关成果荣获中国农药工业协会“技术创新一等奖”，大北农集团“大北农科技奖二等奖”。培养博士后2名，博士研究生1名、硕士研究生2名。按计划完成了各项研究任务，在学术论文、专利和人才培养等指标方面，超额完成。