Cry1Ah蛋白杀虫特异性分子机制

There are 622 cry genes cloned in the world now, but most of them can't be utilized directly because of the low toxicity or narrow insecticidal spectrum. The novel gene cry1Ah, intellectual property rights owned, was cloned by our lab, it encoded a protein exhibited a strong toxicity against many lepidopteran insects, except Bombyx mori. The transgenic maize and rice carrying the cry1Ah gene have been assessed in field trial stage, and it will become the important guarantee for the creation of the new generation biotechnological crops. In this proposal, we are planning to analyze interaction between Cry1A protein and BBMV from insect midgut, and binding difference between Cry1Ah and Cry1Ai, which shares the similar structure but contrary function with Cry1Ah protein, will be investigated. To search the specific binding protein in BBMV, to explore the Loop region of specific insecticidal activity from Cry1Ah/Cry1Ai protein, and to reveal the molecular mechanism of Cry1Ah with strong toxicity against many agricultural insect pests such as Helicoverpa armigera, and safe to economic insect Bombyx mori,will be conducted furthermore.In this proposal, the national significant demand is being aimed, the mechanism of insecticidal specificity of Cry1Ah will be elucidated. All of these research progress will lay the foundation for the promotion and application of cry1Ah gene crops, meanwhile, it will provide the theoretical guidance for the molecular designing and directed modification on cry genes.

目前全球发现的cry基因已达622种，但由于毒力有限或杀虫谱窄等原因，大部分cry基因无法直接应用。Cry1Ah是本实验室克隆的具有自主知识产权的新型杀虫基因，其编码的蛋白对多种鳞翅目害虫具有很高的杀虫活性，且对家蚕毒性很低，目前转cry1Ah基因水稻和玉米已经进入田间试验阶段，成为我国新一代抗虫转基因作物创制的保证。本项目拟以结构相似、杀虫活性"相反"的Cry1Ah和Cry1Ai蛋白为研究材料，分析比较两者与昆虫BBMV的互作情况，寻找昆虫BBMV上特异性结合蛋白，明确Cry蛋白与杀虫特异性相关的Loop区域氨基酸组成，揭示Cry1Ah蛋白对棉铃虫等农业害虫高毒力、对经济昆虫家蚕相对安全低毒的分子机制。这项研究瞄准国家重大需求，不仅从理论层面揭示Cry1Ah蛋白的杀虫特异性机制，为cry1Ah基因的推广应用奠定坚实的理论基础，同时也为其它cry基因的高效、安全分子设计提供理论指导。

Cry1Ah蛋白是我国具有自主知识产权的新型、高效的抗虫蛋白，对棉铃虫、亚洲玉米螟、小菜蛾、水稻二化螟等鳞翅目害虫具有较高的杀虫活性，对重要的经济昆虫家蚕安全无毒。本项研究通过比较Cry1Ah蛋白与棉铃虫、家蚕BBMV的互作分析，明确了杀虫特异性与BBMV特异性结合的关系，利用配体垂钓平台鉴定了Cry1Ah蛋白在棉铃虫BBMV上的特异性结合蛋白——APN1，并确定了棉铃虫APN1上A548至S798区域是APN1与Cry1Ah蛋白互作的区域。本研究以结构相似、功能“相反”的Cry1Ai蛋白为对比材料，比较Cry1Ah蛋白和Cry1Ai蛋白的三维结构和各结构域的氨基酸序列，分析可能影响杀虫特异性的氨基酸区域，并通过体外竞争结合实验，明确了Cry1Ah蛋白Domain II的Loop 2和Loop 3共同影响其杀虫特异性，最后将Cry1Ah蛋白上决定杀虫特异性的氨基酸序列“移植”到Cry1Ai蛋白上，重组的Cry1Ai蛋白获得了对棉铃虫的杀虫活性，从而改变了Cry1Ai蛋白的杀虫特异性。这些结果不仅从分子层面揭示Cry1Ah蛋白的杀虫特异性机制，同时也为转cry1Ah基因水稻和玉米的推广应用奠定坚实的理论基础。对Cry1Ai蛋白的改造拓宽了cry1Ai基因的应用范围，提升了该基因的利用与价值，同时也为其它cry基因的高效、安全分子设计提供理论指导。.本项目完成期间，在国内外学术期刊发表论文8篇，其中应用微生物顶尖刊物Applied and Environmental Microbiology上发表论文3篇，其它SCI源论文3篇；申请发明专利1项。2016年，发明专利《对鳞翅目昆虫高毒力的 Bt cry1Ah 基因及其表达产物（ZL200410009918.9）》荣获第十八届中国专利奖优秀奖。本项目主持人张杰研究员荣获“全国农业科研杰出人才”。培养博士后1名，博士研究生2名、硕士研究生3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。