CAJ | 학술논문

[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。
[목적]이용형광쌍분자호보기술연구대맥황왜병독운동단백（BYDV-PAV,MP）형성이취체적가능성,병연구MP동형이취체여병독운동지간적관계。[방법]장쌍분자형광호보재체중포함다극륭위점、35S계동자급종지자적DNA편단구건도고패수교고적식물표체재체pCAMBIA1300상,연후이BYDV-PAV적전장cDNA위모판,근거GenBank중등기적BYDV-MP적기인서렬설계인물,통과PCR확증득도목적기인편단BYDV-MP,극륭도경과개조적쌍분자형광호보재체pCAMBIA1300-NE、pCAMBIA1300-CE상,득도료중조적쌍분자형광호보재체。전격전화농간균,이용농간균삼투주사기술주사도연초협편,형광현미경하관찰식물체내적단백호작현상。[결과]농간균삼투주사후,2～5d관찰쌍분자형광호작현상,MP단백호작조급정대조조협편산생황색형광,부대조조미유형광현상。[결론]BYDV-MP재식물체내형성동원이취체,해연구결과위진일보심입개전BYDV운동과정화궤리등연구제공료이론의거。
[Objective] The aim was to investigate the dimer formation between the movement proteins（MP）in barely yellow dwarf virus by using the technology of bimolecular fluorescence complementation technology and to further study the relationship between MP homodimerization and viral movement.[Method] The DNA sequence of bimolecular fluorescent complementary vector containing cloning multiple cloning sites,35S promoter and terminator was cloned into the expression vector pCAMBIA1300,which replicates at a higher copy number in E.coli.Then,the BYDV-MP gene fragment was amplified in the presence of the whole BYDV-PAV cDNA sequence as template and the primers designed according to the BYDV-MP gene sequence from GenBank,cloned into the modified bimolecular fluorescent complementary vectors pCAMBIA1300-NE and pCAMBIA1300-CE.The resulting vectors were transformed into Agrobacterium by electroporation method and infiltrated into the tobacco leaf.Protein interactions were observed under fluorescence microscope.[Result] Yellow fluorescence could be viewed in the leaves co-infiltrated with Agrobacterium carrying pCAMBIA1300NE-MP and pCAMBIA1300CE-MP at 2-5 d post-infiltration,while yellow fluorescence could not be observed in negative control groups.[Conclusion] BYDV-MP formed homodimers in plant cells.The results can provide theoretical basis for further in-depth research about the movement process and mechanism of BYDV.