蝎昆虫毒素BmK IT中关键活性位点功能分析及由AcMNPV介导的抗虫机制研究

Insect-selective scorpion neurotoxins display no or less toxicity toward mammals but are specifically active toward insects. These neurotoxins can interact specifically with sodium channel in excitable cell membrane and disorder the metabolism of insects. The high toxicity and strict insect selectivity of these scorpion toxin make it a promising biological insecticide. Recombinant viruses engineered to produce insect-selective neurotoxins from scorpion venom are considered to have great potential for pest control. Based on our preliminary work, the project intend to investigate the mechanism underlying the excitatory insect toxin (BmK IT) mediated insecticidal activity of Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) and its applicability in plant pest control. Methods of entomology, molecular virology, immunology, molecular biology, proteomics, ecology, bio-informatics will be adopted. Firstly, functional study of three key active sites in BmK IT that are respectively associated with receptor affinity,the species specificity of sodium channel and voltage sensor trapping will be carried out. Subsequently, this project will disclose the molecular mechanism of the recombinant virus AcMNPV-BmK IT's capacity in infecting and damaging lepidopteral insect hosts' tissues and cells. The synergistic action in host tissues between BmK IT and insect-resistance proteins, including chitinase, cathepsin and polyhedrin, will be studied. Finally, ecological safety and permanence of recombinant virus AcMNPV-BmK IT in field test environment will be analyzed. Meanwhile, critical data of short-term and lasting insect control population efficiency and mechanism can also be obtained. Besides promoting the study on the relationship between BmK IT's structure and function, improving insecticidal activity of AcMNPV, this project may serve as a strong technological and theoretical mementum for the development of biological insecticide with independent intellectual property. The results of this project have significant value in the scientific control of lepidopteral insect.

蝎昆虫特异性毒素可刺激昆虫可兴奋细胞而造成虫体各系统代谢紊乱，通过重组蝎昆虫特异性素素提高AcMNPV对农业害虫的防治效果是重要的生物防治策略，但AcMNPV介导的蝎昆虫毒素BmK IT的抗虫确切分子机制尚未阐明。本项目拟在揭示BmK IT的抗虫活性的基础上，研究基于BmK IT提高AcMNPV抗虫活性的作用机制。首先，开展BmK IT中推测与受体亲和性、钠通道种属特异性及电压传感相关的三个关键位点的功能分析；其次，探讨重组BmK IT基因的AcMNPV侵染、破坏鳞翅目害虫宿主组织和细胞的分子机制，分析其与几丁质酶、组织蛋白酶、多角体蛋白等抗虫蛋白对宿主各组织的协同作用机制；第三，研究重组BmK IT的病毒的生态安全性及在中试环境下控制田间害虫种群效能的作用机制。项目成果可深化对BmK IT的结构与功能的认识，并为有效利用其改造AcMNPV提供理论依据，对科学防治鳞翅目害虫具有重要意义。

蝎昆虫特异性毒素可刺激昆虫可兴奋细胞而造成虫体各系统代谢紊乱，通过重组蝎昆虫特异性素素提高AcMNPV对农业害虫的防治效果是重要的生物防治策略。本项目在揭示BmK IT的抗虫活性的基础上，研究基于BmK IT提高AcMNPV抗虫活性的分子作用机制。首先，开展了BmK IT中推测与受体亲和性、钠通道种属特异性及电压传感相关的三个关键位点的功能分析，明确了关键位点对宿主细胞的作用及种属特异性的影响。其次，开展了重组BmK IT的AcMNPV侵染鳞翅目害虫宿主的机理及不同抗虫基因的协同作用研究，明确了重组病毒抗虫的过程和规律；分析了BmK IT在虫体不同组织的表达情况；证实了不同虫龄的棉铃虫幼虫对重组病毒敏感性的差异；分析了AcMNPV早、晚期启动子调控表达BmK IT对棉铃虫体液免疫的影响；鉴定出一株高抗虫活性的重组病毒株。第三，分析了重组BmK IT的AcMNPV在宿主细胞Sf9内的作用机制。开展了AcMNPV和AcMNPV-BmK IT(IE1)侵染Sf9细胞在转录组水平的高通量测序和分析，相关数据提交至NCBI SRA库（编号为：SRP075752, SRP075795, SRP075799），筛选和鉴定了一系列受BmK IT表达调控的病毒和宿主细胞的蛋白因子；明确了AcMNPV介导表达的BmK IT及其它外源蛋白对宿主细胞凋亡及对病毒复制的影响；分析了杆状病毒的凋亡抑制蛋白Ac-IAP1和Ac-IAP2在病毒入侵宿主细胞过程中的效应；证实了重组BmK IT病毒对宿主细胞参与凋亡的相关基因以及对AcMNPV中重要蛋白38K、F、C42、P78转录水平的影响；分析了BmK IT对病毒与宿主细胞骨架(F-actin)的互作和重组病毒在细胞内运输的影响。第四，进行了重组BmK IT的AcMNPV的规模化制备研究，开展了重组病毒的动物安全性评价及AcMNPV控制田间害虫种群效能的作用分析。本项目共计发表学术论文22篇（其中SCI 收录15篇），申报国家发明专利一项（病毒专利可应用于植保、昆虫细胞表达等）。项目结果从细胞、虫体、生态三个层次对重组BmK IT的AcMNPV的抗虫作用机制作了系统分析，为有效利用改造AcMNPV提供依据，对科学防治鳞翅目害虫有重要意义。