双分子荧光互补技术(BiFC)分析玉米SSⅠ与PPDK1之间的蛋白互作

Interactions between SSⅠ and PPDK1 of maize using bimolecular fluorescence complementation (BiFC) assay

【目的】分析玉米胚乳可溶性淀粉合成酶(SSⅠ)与质体型糖酵解途径关键催化酶丙酮酸磷酸双激酶(PPDK1)的蛋白互作关系,揭示可能发生互作的亚细胞位置。【方法】采用酶切连接的方法构建326-CYCHA-ss1和326-CYNEE-ppdk1双分子荧光互补表达载体,转化农杆菌EHA105,瞬时浸染烟草叶片组织,激光共聚焦显微镜下观察SSⅠ和PPDK1相互作用产生的荧光信号。【结果】双酶切试验鉴定表明,326-CYCHA-ss1和326-CYNEE-ppdk1重组载体构建正确;PCR结果证实,植物表达载体成功转化到农杆菌EHA105中;双分子荧光互补试验中,可观测到SSⅠ和PP-DK1相互结合而产生的黄色荧光信号。【结论】证实SSⅠ和PPDK1能够在植物活体细胞内发生真实的蛋白互作。

【Objective】 Protein interactions between soluble starch synthase（SSⅠ） and acetone phosphate double kinase（PPDK1） of the glycolytic pathway in maize endosperm were analyzed to reveal the location of interactional subcellular.【Method】 Enzyme digestion and ligation method was used to construct the expression vector of 326-CYCHA-ss1 and 326-CYNEE-ppdk1 and the constructed expression vector was transformed into Agrobacterium tumefaciens EHA105,which was used to dip tobacco leaf tissue momentarily.At last the interactions of SSⅠ and PPDK1 were observed using confocal laser scanning microscope.【Result】 Results showed that 326-CYCHA-ss1 and 326-CYNEE-ppdk1 vectors were constructed and expressed successfully.Yellow fluorescent BiFC signals expressed strongly in vivo.【Conclusion】 Real interactions between SSⅠ and PPDK1 were observed in living cells.