Alphasatellite伴随双生病毒病害复合体的生物学功能研究

Alphasatellites are circular single-stranded molecules associated with begomovirus / betasatellite complexes. Studies on alphasatellites mainly focus on molecular characterization, while the function of alphasatellites is not known. Our preliminary work showed the types and molecular variation of alphasatellites in China. In this project, the post-transcriptional gene silencing (PTGS) and transcriptional gene silencing (TGS) suppressor activity of the Rep protein encoded by the alphasatellite were tested. In order to evaluate the interaction among the alphasatellite, begomovirus and betasatellite, the infectious clones of begomovirus, alphasatellite and betasatellite molecules of typical geminiviruses will be constructed and the influence of alphasatellite in viral pathogenicity will also be analyzed. To assess the interaction between the alphasatellite and the host, the host factor will be screened, and the begomovirus / alphasatellite / betasatellite complexes will be agro-infected in host factor silenced-plants or over expressed-plants, and the function of alphasatellite in plant defense will be studied. The difference of whiteflies acquisition, retention and inoculation in alphasatellite-infected or no alphasatellite-infected plants will be compared to dertermine the influence on alphasatellite in virus transmission. With these above research work, it will expand our knowledge of the functions of alphasatellite associated with begomovirus / betasatellite complexes in viral pathogenicity, plant defense and virus transmission.

Alphasatellite总是与双生病毒病害复合体相伴随，但目前对alphasatellite的研究仍集中在田间的分离鉴定，其生物学功能不清。项目拟以与双生病毒病害复合体伴随的alphasatellite为对象，在完善核酸数据库的基础上，明确其编码的唯一蛋白Rep抑制PTGS和TGS的功能；构建双生病毒各DNA分子的侵染性克隆，分析其对双生病毒致病及病毒DNA、卫星beta积累量的影响，明确其与双生病毒各DNA分子之间的互作。筛选可能与Rep互作的寄主因子，沉默或过表达寄主因子后接种病毒，观察其对病毒致病及植物防御途径的影响，探索其与寄主植物的互作。比较烟粉虱在有/无卫星alpha的植物上获毒、持毒及传毒效率的差异，明确alphasatellite对介体传毒能力的影响。项目的开展将明确alphasatellite在双生病毒病害复合体致病、植物防御反应及介体传播中的生物学功能。

Alphasatellites是一类单链环状DNA分子，编码唯一蛋白Rep。在我国，alphasatellite常与双生病毒病害复合体begomovirus/betasatellite共同侵染作物和杂草，造成严重经济损失。目前，对alphasatellite的研究主要集中在田间的分子鉴定，而对其生物学功能研究较少。本项目以我国主要发生的3类alphasatellite分子为代表，将其编码的Reps分别转化重组表达载体，浸润转GFP基因的16c或16-TGS本氏烟，经紫外灯下观察、Northern blot及Western blot分析，明确其编码的Rep蛋白能抑制转录后和转录水平基因沉默。对alphasatellite与同源或异源辅助病毒/betasatellite的互作发现，当alphasatellite存在时，同源或异源辅助病毒及伴随的betasatellite的积累量降低，降低程度受不同辅助病毒和alphasatellite的影响。酵母双杂交发现alphasatellite-Rep只与辅助病毒编码的Rep蛋白互作，而与其他蛋白(AV1-AV2，AC2-AC4)及 betasatellite编码的βC1均不互作。利用转录组高通量测序技术筛选与alphasatellite互作的寄主因子，TYLCCNV/TYLCCNB 和 TYLCCNV/TYLCCNB/TYLCCNA组合接种的本氏烟中分别鉴定到2272 和1207差异表达基因 (DEGs) 。经GO功能和KEGG富集分析，并结合DEGs的差异倍数，筛选出对应于alphasatellite的36个DEGs。VIGS技术分别沉默其中6个DGEs，再进行病毒攻毒接种。与沉默GFP后的对照组相比，沉默靶标基因的植株发病时间均提前，症状更为严重，且接种TYLCCNV/TYLCCNB/TYLCCNA的比接种TYLCCNV/TYLCCNB的植株发病时间延迟，症状稍微减弱。qPCR和southern blot也得到相同结果。这说明这些寄主因子沉默后本氏烟对双生病毒的侵染更加敏感，暗示这6个DGEs与寄主植物抗病相关。Alphasatellite可能通过与这些寄主因子互作以增强寄主植物对TYLCCNV/TYLCCNB的防御能力，进而负调控TYLCCNV/TYLCCNB病害复合体对寄主植物造成的危害。