[目的]研究番茄斑萎病毒(Tomato spotted wilt virus,TSWV)运动蛋白NSm的细胞定位.[方法]将NSm基因融合GFP后构建到植物双元表达载体pCHF3中,农杆菌介导浸润本氏烟叶片,同时将融合GFP的NSm基因利用Bac-to-Bac杆状病毒表达体系转染昆虫Tn细胞.在激光共聚焦显微镜下观察NSm-GFP在烟草表皮细胞和昆虫Tn细胞中的定位情况.[结果]观察发现与单独表达GFP在细胞壁周围和细胞核处均匀分布不同,NSm-GFP融合蛋白会在植物细胞内扩散,能够在细胞壁边缘定位,并且在胞间连丝处呈不连续的绿色荧光小点,偶尔成对出现在相邻的两个细胞之间；NSm蛋白在昆虫Tn细胞表面产生数量众多的管状结构并向外延伸.[结论]研究结果表明NSm能够特异性定位在植物细胞的胞间连丝处,并能在昆虫Tn细胞内表达,在细胞表面产生运动蛋白小管.%[Objective] Expression and subcellular location of NSm protein of Tomato spotted wilt virus were studied using plant and insect cells.[Methods] First, the NSm gene, located on the ambisense M RNA segment of tomato spotted wilt virus, was cloned into the pCHF3 vector which includes a GFP gene.Agrobacterium-mediated transient expression from N.benthamiana leaves was used to study the location of NSm in plant cells.Second, to test whether plant-specific components were involved in tubule formation, the NSm gene was also expressed in a heterologous expression system, i.e., insect cells.T.ni (Tn) cells were infected with a recombinant baculovirus expressing the NSm gene.[Results] NSm-GFP fusion proteins diffused in the tobacco epidermal cells and were located at the edge of the cell walls.These proteins can also form discontinuous green fluorescent spots at the plasmodesmata, which were sometimes present in pairs between two neighboring cells.However, GFP proteins expressed alone distributed evenly around the cell wall and in the nucleus.In the entomic Tn cells, NSm proteins formed a large number of tubular structures extending from the surface.[Conclusion] These findings suggest that NSm protein target the plasmodesmata specifically in plant cells, and they also could form tubular structures on the surface when expressed in entomic Tn cells.
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