烟芽夜蛾囊泡病毒3h株干扰宿主细胞周期的分子机制

To discover and develop any potential efficient pathogenic microorganisms as biological-pesticide is one of the most urgent requirement in insect pest bio-control. As the first reported ascovirus isolated from China, the high virulent strain Heliothis virescens ascovirus 3h (HvAV-3h) would be studied in this project. The effects of HvAV-3h infection on the host cell cycle (Spodoptera exigua fat body cell, SeFB) will be detected by flow cytometry. The data collected from transcriptome would be used to select cyclins encoded by Spodoptera exigua, and the RNAi will be performed to validate the function of those selected cyclins. The key cyclins would be cloned and expressed to prepare polyclonal antibody, and immunofluorescence observation will be performed to detect the key cyclins’ expression pattern. Several early proteins encoded by HvAV-3h would be selected and used to test the interaction with cyclins in the further experiments. At last, the mechanism of host cell cycle alteration by the infection of HvAV-3h would be declined. All the results in this study will lay the foundation for elucidating the specific “acute pathogenicity” mechanism of HvAV-3h, and provide scientific basis for the popularization and application of pest biological control.

深入挖掘潜在的高致病性病原微生物资源是当前害虫生物防治发展的重大需求之一。本项目以分离自我国本土的高致病性烟芽夜蛾囊泡病毒3h株（HvAV-3h）为研究对象，利用流式细胞仪检测HvAV-3h感染所致的甜菜夜蛾脂肪体细胞系（SeFB）细胞周期进程的变化；基于转录组数据对甜菜夜蛾编码的细胞周期蛋白进行表达差异分析，结合RNA干扰技术对不同细胞周期蛋白的功能进行验证；对起关键作用的细胞周期蛋白基因进行克隆、表达、制备抗体，通过免疫荧光观察明确关键细胞周期蛋白的表达时空。同时，对HvAV-3h编码的早期表达蛋白进行筛选，挑选对宿主细胞周期具有显著影响的早期表达蛋白，深入研究其与宿主细胞间的互作关系，最终明确HvAV-3h感染对宿主细胞周期影响的分子机制。本项目研究结果将为阐明HvAV-3h感染导致“急性致病”的分子致病机制奠定基础，进一步为HvAV-3h在害虫防控实践中的推广应用提供科学依据。

HvAV-3h的感染诱导SeFB细胞周期停滞于G2/M期。流式细胞仪检测结果表明健康的SeFB细胞的细胞周期时相分布为G1 = 29.52 ± 1.10%，S = 30.33 ± 1.19%和G2/M = 40.06 ± 0.75%。细胞周期抑制剂羟基脲（Hydroxyurea，HU）同步SeFB细胞于G1/S期后，流式细胞仪测定结果表明细胞培养倍增时间约为24 h，其中G1→S、S→G2/M、G2/M→G1时长分别约为8 h。HvAV-3h的感染诱导SeFB细胞周期停滞于G2/M期。用流式细胞仪分别检测HvAV-3h感染后不同时间点的SeFB细胞，发现病毒感染12 h后，60%以上的SeFB细胞被抑制在G2/M期，G1期细胞占比不到10%。HvAV-3h感染同步于G1/S期的SeFB细胞，流式细胞仪检测结果表明病毒感染后24 h，70%左右的细胞被抑制在G2/M，G1期细胞占比不到2%。HvAV-3h感染对宿主细胞Cyclin B1及CDK1转录水平的影响。转录组数据深入分析，筛选得到6个甜菜夜蛾Cyclin基因和1个CDK1基因。经过实时荧光定量PCR验证发现，囊泡病毒感染显著抑制Cyclin B1，Cyclin B2，Cyclin H的转录，但对Cyclin B3，Cyclin A和Cyclin D的转录无明显影响；在囊泡病毒感染3 h后，宿主细胞的CDK1转录水平均低于健康组细胞中CDK1的转录水平。HvAV-3h感染对宿主细胞Cyclin B1及CDK1表达水平的影响。利用获得的Cyclin B1和CDK1抗血清进行Western blotting分析，结果表明SeFB细胞被HvAV-3h感染后24 h内均可检测Cyclin B1和CDK1表达，且在病毒感染后12-24 h，Cyclin B1和CDK1的表达量均被抑制。