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[目的]利用荧光双分子互补技术研究大麦黄矮病毒运动蛋白(BYDV-PAV,MP)形成二聚体的可能性,并研究MP同型二聚体与病毒运动之间的关系。[方法]将双分子荧光互补载体中包含多克隆位点、35S启动子及终止子的DNA片段构建到拷贝数较高的植物表达载体pCAMBIA1300上,然后以BYDV-PAV的全长cDNA为模板,根据GenBank中登记的BYDV-MP的基因序列设计引物,通过PCR扩增得到目的基因片段BYDV-MP,克隆到经过改造的双分子荧光互补载体pCAMBIA1300-NE、pCAMBIA1300-CE上,得到了重组的双分子荧光互补载体。电击转化农杆菌,利用农杆菌渗透注射技术注射到烟草叶片,荧光显微镜下观察植物体内的蛋白互作现象。[结果]农杆菌渗透注射后,2～5d观察双分子荧光互作现象,MP蛋白互作组及正对照组叶片产生黄色荧光,负对照组未有荧光现象。[结论]BYDV-MP在植物体内形成同源二聚体,该研究结果为进一步深入开展BYDV运动过程和机理等研究提供了理论依据。
[Objective] The research aimed to study the possibility of formation of dimers of barley yellow dwarf virus (BYDV-PAV, MP) by fluorescent double-molecule complementation technique and to study the relationship between MP homodimers and virus movement. [Method] The DNA fragment containing the multiple cloning site, 35S promoter and terminator in the bimolecular fluorescent complementary vector was constructed on the plant expression vector pCAMBIA1300 with higher copy number. Then the full-length cDNA of BYDV-PAV was used as a template, According to the gene sequence of BYDV-MP registered in GenBank, the target gene fragment BYDV-MP was amplified by PCR and cloned into the modified double-molecule fluorescent complementary vector pCAMBIA1300-NE, pCAMBIA1300-CE to obtain recombinant double Molecular fluorescence complementary carrier. Agrobacterium tumefaciens was electroporated and transformed into tobacco leaves by Agrobacterium tumefaciens injection technique. The protein interactions in plants were observed under a fluorescence microscope. [Result] The bimolecular fluorescence interaction was observed 2 ~ 5 days after Agrobacterium tumefaciens injection. The yellow fluorescence was produced in leaves of MP protein interaction group and positive control group, but no fluorescence was observed in negative control group. [Conclusion] BYDV-MP formed homodimers in plants. The results of this study provide a theoretical basis for further study on the movement process and mechanism of BYDV.