PVX病毒载体介导2种马铃薯Y病毒科病毒VPg蛋白表达诱导烟草叶片系统坏死的研究

芜菁花叶病毒（Turnip mosaic virus, TuMV）和槟榔坏死环斑病毒（Areca plam necrotic ringspot virus, ANRSV）是马铃薯Y病毒科（Potyviridae）中分别可诱导十字花科作物和槟榔产生坏死病症的2种不同属病毒。VPg（Viral protein genome linked）作为与马铃薯Y病毒科病毒基因组5′端的结合蛋白,在病毒复制、翻译和运动等侵染过程中发挥重要作用。本研究利用In-Fusion克隆策略分别构建了可表达3′端融合Strep蛋白标签序列的ANRSV和TuMV VPg全长编码基因的马铃薯X病毒（Potato virus X, PVX）载体pPVX-VPg-AR和pPVX-VPg-Tu。通过农杆菌渗透注射接种本生烟（Nicotiana benthamiana）,利用RT-PCR、Strep蛋白标签亲和层析和Western blot等方法证明了PVX病毒载体能够介导VPg-AR和VPg-Tu蛋白在本生烟中系统表达,且发现这2种VPg蛋白的过量表达诱导本生烟叶片产生了不同程度的系统性坏死病症。进一步采用DAB和NBT染色,在产生坏死症状叶片中检测到大量活性氧（reactive oxygen species, ROS）的积累。因此,推测VPg可能是TuMV和ANRSV诱发植物病症产生的一个决定因子,为后续研究这2种病毒诱导症状形成机制奠定了基础。

Expression of the TuMV or ANRSV-encoded VPg Protein Using a Potato Virus X (PVX) Vector Induced Necrotic Symptoms in Nicotiana benthamiana

VPg (viral protein genome-linked) is a protein that is covalently attached to the 5′ end of positive strand viral RNA and acts as a primer during RNA synthesis in Potyviridae. Turnip mosaic virus (TuMV, genus Potyvirus) and Areca plam necrotic ringspot virus (ANRSV, genus Arepavirus) are typical members of in the family Potyviridae and cause damaging disease in many kinds of Brassicaceae and Areca catechu, respectively. In this study, we constructed Potato virus X (PVX) -based vectors pPVX-VPg-AR and pPVX-VPg-Tu for individually expressing TuMV, ANRSV- encoded VPg protein (VPg-AR and VPg-Tu) with a Strep-tag in Nicotiana benthamiana plants by agroinfiltration. The expression of recombinant VPg-AR and VPg-Tu was identified using RT-PCR of total RNA and western blot analysis of strep-tagged proteins from PVX-VPg-AR and pPVX-VPg-Tu -infected plant leaves, respectively. The ectopic expression of VPg-AR and VPg-Tu caused severe systemic necrosis in N. benthamiana. Further study revealed that the overexpression of VPg-AR and VPg-Tu resulted in accumulation of reactive oxygen species (ROS) after 3,3′-diaminobenzidine (DAB)-HCl and nitroblue tetrazolium (NBT) staining for detection of H₂O₂ and O^2- radicals in PVX-VPg-AR and PVX-VPg-Tu-infected plant leaves. The results demonstrated that VPg might be a pathogenicity determinant that plays key roles in symptom formation of TuMV and ANRSV.