适用于板栗疫病菌分子相互作用研究的双分子荧光互补系统

A Bimolecular Fluorescent Complementation System Suitable for Molecular Interaction in Cryphonectria parasitica

双分子荧光互补技术近年来已广泛应用于研究体内蛋白质相互作用。为建立适合于板栗疫病菌的体内蛋白质相互作用检测体系,本研究以质粒pCPXHY2和pCPXG418为骨架,构建了由板栗疫病菌pgd启动子控制的、以增强型黄色荧光蛋白EYFP为报告基因和以潮霉素和G418抗性为选择标记的双质粒系统pCPXHY2N155和pCPXG418C156。质粒pCPXHY2N155携带EYFP的1~155位氨基酸残基的编码序列,pCPXG418C156携带EYFP基因的156~239位氨基酸残基的编码序列。为验证该质粒系统的有效性,将板栗疫病菌异质三联体G蛋白的β和γ亚基基因分别与pCPXHY2N155上的N155和pCPXG418C156上的C156相连,获得重组质粒pCPXHY2N155β和pCPXG418C156γ。将这两个重组质粒共转化板栗疫病菌野生型菌株EP155,在转化株中观察到明显的黄色荧光,表明Gβ和Gγ在细胞中发生了相互作用。本研究所构建的双分子荧光互补系统,为今后研究板栗疫病菌分子相互作用提供了新的技术手段。

Bimolecular fluorescence complementation system has been widely used to study protein-protein in- teraction in vivo. To establish a detection system for protein-protein interaction suitable for chestnut blight fun- gus, a complementation system composed of constructs pCPXHY2N155 and pCPXG418C156 which were boned on plasmids pCPXHY2 and pCPXG418 with a pgd promoter and EYFP as reporter and hygromycin and G418 as selection markers, respectively, were constructed. The coding sequence for 1-155 amino acids of EYFP with a multicloning site linker was inserted into the pCPXHY2N155, and the coding sequence for 156-239 amino acids of EYFP with a multicloning site linker was inserted into the pCPXG418C156. The heterologous trimeric G-pro- tein subunits Gβ and Gy in C. parasitica were used to test the effectiveness of the system. The coding sequences for Gβ and Gy were cloned into pCPXHY2N155 and pCPXG418C156, respectively, to give rise to plasmids pCPXHY2N 155 β and pCPXG418C 156γ Co-transformation of C. p armitic a wild-type strain EP 155 with pCPX- HY2N 155 β and pCPXG418C156γ resulted in yellow fluorescence in the hyphae of the transgenic strains, indicat- ing that the interaction of Gβ and Gy had occurred in vivo. The availability of the bimolecular fluorescence com- plementation system provides a new tool for observing the protein-protein interaction in C. parasitica.