病毒介导的GFP-ATG8在小麦上的表达和在自噬活性监测上的应用

Virus-mediated expression of GFP-ATG8 for autophagy monitoring in wheat

自噬相关因子ATG8定位于自噬结构的膜上,荧光蛋白标记的ATG8在过表达细胞内所呈现的点状荧光常用于表征自噬结构和监测自噬活性。病毒介导的过表达(virus-mediated over-expression,VOX)是一种简便、快速制备目的基因过表达植株的技术。采用基于狗尾草花叶病毒FoMV的VOX技术(FoMV-VOX)在小麦植株中表达GFP标记的小麦ATG8家族成员TaATG8a,建立小麦活体植株的自噬活性监测技术平台。构建了融合基因GFP-TaATG8a的FoMV-VOX载体,采用Agroinfiltration方法在本氏烟草叶片中表达携带GFP-TaATG8a的FoMV基因组RNA和组装病毒粒子,将烟草汁液中的病毒粒子摩擦接种于小麦幼苗植株叶片,对接种植株叶片和根组织中的荧光信号进行观察和特征鉴定。结果表明,采用FoMV-VOX技术在小麦植株上不仅可以实现GFP-TaATG8a在接种叶片中的高效表达,还可以借助病毒的系统侵染实现该融合基因在未接种叶片和根组织中的高效表达。经饥饿处理激活自噬,融合蛋白GFP-TaATG8a在植株叶表皮、叶肉以及根细胞中呈现表征自噬结构的点状荧光。采用FoMV-VOX技术获得的GFP-TaATG8a过表达植株可以应用于小麦多种组织类型中的自噬活性调节机制和生理功能研究。

ATG8 is an essential autophagy-related factor decorating on the membranes of autophagic structures.Fluorescence protein-tagged ATG8 expressed in live cells has been widely used to visualize autophagic structures and to monitor the activity of autophagy.Virus-mediated over-expression(VOX)is a simple technique for rapid expression of genes of interest in plants.Here the foxtail mosaic virus(FoMV)-based VOX was adopted for preparation of wheat seedlings over-expressing the GFP-tagged form of the wheat ATG8 family member TaATG8a.An FoMV-VOX vector was constructed for expression of the recombinant FoMV genomic RNA carrying the GFP-TaATG8a sequence.Expression of FoMV genomic RNA and assembly of FoMV virions were accomplished in Nicotiana benthamiana leaves through agroinfiltration.N.benthamiana leave extract containing FoMV virions was used to inoculate leaves of wheat seedlings.Fluorescence microscopy of virus-inoculated wheat seedlings showed the efficient expression of GFP-TaATG8a was in not only inoculated leaves but also systemic uninoculated leaves and roots.More-over,punctate fluorescence of GFP-TaATG8a representing autophagic structures was clearly observed in leaf epidermal cells,mesophyll cells,and root cells of wheat seedlings subjected to autophagy-stimulating starvation stress.The autophagy activity in these cells could be evaluated by quantifying the GFP-TaATG8a-labeled autophagic structures.These results lay a foundation for studies of the regulating mechanisms and physiological roles of autophagy in various wheat tissues.