玉米AGPase-bt2与GPN1蛋白互作的双分子荧光互补技术研究
Interaction between AGPase-bt2 and GPN1 in maize using bimolecular fluorescence complementation

【目的】解析玉米淀粉合成限速酶ADP 葡萄糖焦磷酸化酶（AGPase-bt2）与糖酵解关键酶甘油醛-3-磷酸脱氢酶（GPN1）在植物体内是否存在相互作用。【方法】首先克隆获得AGPase-bt2和GPN1基因，然后采用双分子荧光互补技术（BiFC），构建326-CYCHA-AGPase-bt2和326-CYNEE-GPN1双分子荧光表达载体，转化农杆菌EHA105，并用2种载体共同瞬时侵染烟草叶肉细胞，48 h后在激光共聚焦显微镜下观察AGPase-bt2与GPN1的相互作用。【结果】AGPase-bt-2基因长1 428 bp，GPN1基因长1 497 bp。双酶切鉴定结果表明，326-CYCHA-AGPase-bt2、326-CYNEE-GPN1载体构建正确；PCR结果证实，植物表达载体成功转化到农杆菌EHA105中；携带有共转化基因的烟草叶肉细胞在激光共聚焦显微镜下观察到明显的黄色荧光现象，且黄色荧光与叶绿体自发的红色荧光位置重合。【结论】AGPase-bt2与GPN1在植物细胞中真实地存在蛋白互作。
【Objective】This study aimed to clarify the interaction between starch synthesis rate-limiting enzyme ADP-glucose pyrophosphorylase (AGPase-bt2) and glycolytic key enzyme glyceraldehyde-3-phosphate dehydrogenase (GPN1) in maize.【Method】 AGPase-bt2 and GPN1 gene was cloned first.Bimolecular fluorescence complementation（BiFC） was used to construct the bimolecular fluorescence expression vectors 326-CYCHA-AGPase-bt2 and 326-CYNEE-GPN1 and they were transformed into Agrobacterium tumefaciens EHA105.Then two vectors were used to dip tobacco mesophyll cells momentarily and the interaction between AGPase-bt2 and GPN1 was observed by using confocal laser scanning microscope after 48 hours.【Result】AGPase-bt2 gene length is 1 428 bp,and GPN1 gene length is 1 497 bp.The 326-CYCHA-AGPase-bt2 and 326-CYNEE-GPN1 vectors were constructed correctly by double enzyme digestion and expressed successfully by PCR.The tobacco mesophyll cells carrying co-transformed gene can be observed as yellow fluorescence phenomenon by laser confocal microscopy,and the yellow fluorescence coincided with spontaneous red fluorescence of chloroplasts.【Conclusion】The interaction between AGPase-bt2 and GPN1 in plant cells was confirmed