烟芽夜蛾囊泡病毒3h株对甜菜夜蛾脂肪体细胞的抗凋亡反应机制

Ascovirus is a group of insect specific double-stranded DNA virus with a great potential to develop into commercial bio-pesticide. As the first ascovirus isolates reported in our country, the Heliothis virescens ascovirus 3h (HvAV-3h) owns the typical “acute pathogenic” and “chroniclethal” phenotype with strong virulence. In this research, the hydrogen peroxide will be used as inducer to induce the apopotosis of host cells, and MTT colorimetric method, confocal laser scanning microscope observation, and flow cytometry analysis would be performed to investigate the anti-apopotosis ability of HvAV-3h. In order to further investigate the key gene in the anti-apopotosis processes, the RNA-seq will be performed with the RNA samples extracted from the treated cells at the key time of point selected from above, and the apopotosis associated DGEs (different expressed genes) would be confirmed by RNA interference. Finally, the selected “anti-apopotosis acting factor” and “apopotosis acting element” would be cloned, prokaryotic expressed, and eukaryotic expressed in vitro to confirm their function via immunofluorescence observation, respectively. All the results we get in this research would provide theoretical foundation for the further investigation of ascovirus pathogenic mechanism.

囊泡病毒是一类专性寄生于昆虫体内的大型双链DNA病毒，具有强大的生物杀虫剂发展潜力。烟芽夜蛾囊泡病毒3h株（Heliothis virescens ascovirus 3h, HvAV-3h）是我国首次分离并报道的本土高毒力致病毒株，具有典型的“急性致病”和“慢性致死”致病特征。本研究将以HvAV-3h为研究对象，以双氧水作为外界的细胞凋亡诱导因子，通过MTT比色法、流式细胞观察等研究方法，全面检测HvAV-3h在甜菜夜蛾脂肪体细胞系中的抗凋亡能力。对筛选出来的关键作用时期，以转录组测序和RNA干扰等技术手段，对细胞凋亡的相关差异基因进行筛选和验证。最后，利用基因克隆、蛋白的原核及真核表达、免疫荧光观测等分子生物学手段，对筛选得到的关键“抗凋亡因子”和“凋亡作用元件”进行单独的体外表达并进行功能验证，阐明HvAV-3h的抗凋亡机制。

囊泡病毒是一类DNA病毒，主要感染鳞翅目害虫的幼虫。本项目主要以离体培养的甜菜夜蛾脂肪体细胞系（SeFB）为对象，展开了烟芽夜蛾囊泡病毒3h（HvAV-3h）株感染后对宿主细胞抗凋亡机制的研究。首先，我们通过化学试剂诱导凋亡的形式对比了健康SeFB和囊泡病毒感染的SeFB细胞对化学试剂诱导凋亡的抵抗能力差异；其次，我们通过转录组、蛋白组和代谢组的测序对囊泡病毒的感染以及化学试剂诱导的囊泡病毒感染的细胞中的大数据进行了获取和对比；再者，我们针对囊泡病毒的一个特殊蛋白——caspase-like蛋白进行了详细的功能研究，发现了其可能对宿主凋亡进程有存在特殊的调控机制——显性负性调控机制；而且，我们根据获得的转录组、蛋白组等数据，对囊泡病毒感染中的重要信号转导蛋白进行了功能分析；最后，我们还对囊泡病毒的安全性进行了详细的分析，为囊泡病毒后期作为生物防治微生物的开发和应用奠定了基础。本项目共资助发表SCI论文6篇，待发表2篇。培养硕士研究生4名，其中2名已经取得硕士学位。