白桦开花抑制因子BpFLC与BpSVP蛋白互作的结构域筛选与互作验证

Identification of Protein Interactions between Flowering Repressors BpFLC and BpSVP from Betula platyphylla

为深入研究白桦开花抑制因子FLC与SVP的相互作用的分子机理。从重组质粒pGBKT7-BpFLC、pGBKT7-BpSVP分别克隆出6个BpFLC截短体（BpFLC1～6）和6个BpSVP截短体（BpSVP1～6），分别编码MI、MIK、K、IKC、KC和C域。在酵母Y2HGold菌中，分别共转诱饵质粒pGBKT7-BpFLC1～6×pGADT7-BpSVP及pGBKT7-BpFLC×pGADT7-BpSVP1～6。酵母转化子Y2HGold[pGBKT7-BpFLC2～5×pGADT7-BpSVP]，可在选择性固体培养基TDO、QTO／A上生长，并在QDO／A／X上长出蓝色菌落，表明BpSVP能与截短体蛋白BpFLC2～5异源结合。此外酵母Y2HGold[pGBKT7-BpFLC×pGADT7-BpSVP2～5]也能同时激活报告基因AUR1-C、HIS3、ADE2、MEL1。进一步研究发现：Y2HGold[pGBKT7-BpFLC3×pGADT7-BpSVP3]存在相互作用，表明BpFLC的K域与BpSVP的K域能够异源结合，是介导BpFLC与BpVP蛋白互作的关键结构域。

The experiment was conducted to study the mechanism of interaction between SVP and FLC in Betula platyphylla. The truncated genes of BpFLC1 -6 and BpSVP1 -6 were, respectively, cloned from yeast recombination plasmids pGBKT7-BpFLC and pGBKTT-BpSVP, pGBKT7-BpFLC1 - 6 × pGADT7-BpSVP and pGBKT7-BpFLC × pGADT7-BpSVP1 - 6 were co-transferred into yeast Y2HGold. The yeast strains of Y2HGold [ pGBKT7-BpFLC2 -5 × pGADT7-BpSVP] could grew on selective agar plates TDO, QTO/A and QDO/X/A with blue stains. BpSVP truncated forms and BpFLC2 -5 protein could act with each other to form heterodimers. Then, Y2HGold[ pGBKT7-BpFLC× pGADT7-BpSVP2 -5 ] were brought into proximity to form protein compounds and activate transcription of four independent reporter genes of AUR1-C, HIS3, ADE2 and MEL1. The interactions between BpFLC3 and BpSVP3 were tested to confirm the acting domains. The yeast Y2HGold [ pGBKT7-BpFLC3 × pGADT7-BpSVP3 ] exhibited blue stains on selective agar plates QDO/X/A. The K domain of BpFLC and that of BpSVP were the key structure domains and mediated the protein interactions between BpFLC and BpSVP.