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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。
In recent years, bimolecular fluorescence complementary technology has been widely used to study the protein interactions in vivo. In order to establish an in vivo protein interaction assay system suitable for T. bungeana, the plasmid pCPXHY2 and pCPXG418 were used as the framework to construct the pgd promoter controlled by the pusillus thuringiensis, and the enhanced yellow fluorescent protein EYFP was reported as the reporter gene. Mycotoxin and G418 resistance are the selectable marker dual plasmids pCPXHY2N155 and pCPXG418C156. Plasmid pCPXHY2N155 carries the coding sequence of amino acid residues 1 to 155 of EYFP and pCPXG418C156 carries the coding sequence of amino acid residues 156 to 239 of EYFP gene. To verify the availability of this plasmid system, the β and γ subunits of the G protein of T. heterostreae were ligated with N155 on pCPXHY2N155 and C156 on pCPXG418C156 to obtain the recombinant plasmids pCPXHY2N155β and pCPXG418C156γ, respectively. The two recombinant plasmids were co-transformed into the wild type strain EP155 of Phytophthora Bl., And the obvious yellow fluorescence was observed in the transformants, indicating that Gβ and Gγ interacted in the cell. The bimolecular fluorescence complementary system constructed in this study provides a new technical means for the future study of the molecular interaction of chestnut blight bacteria.