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为阐明芥菜开花路径核心调节子SVP与FLC相互作用的结构域,从酵母重组质粒pGADT7SVP、pGBKT7FLC分别亚克隆了5个SVP截短体(SVP1～5)和5个FLC截短体(FLC1～5)。SVP1～5与FLC1~5编码蛋白的结构域均分别为MI、MIK、K、IKC和KC。利用酵母双杂交体系,分别构建酵母猎物质粒pGADT7SVP1～5与诱饵质粒pGBKT7FLC1～5,并转化对应的酵母Y187、Y2HGold菌。酵母转化子Y187[pGADT7SVP2～5]能与Y2HGold[pGBKT7FLC]融合,并可在选择性固体培养基QDO/X/A上长出蓝色菌落,表明FLC能与截短体蛋白SVP2～5异源结合,SVP的K域(SVP3)可独立作用于FLC蛋白。此外,Y187[pGADT7SVP]×Y2HGold[pGBKT7FLC2～5]也能同时激活报告基因AUR1-C、HIS3、ADE2、MEL1,表明FLC的K域(FLC3)也可独立作用于SVP。进一步研究发现:Y187[pGADT7SVP3]×Y2HGold[pGBKT7FLC3]正向杂交以及Y187[pGADT7FLC3]×Y2HGold[pGBKT7SVP3]载体互换后杂交均可相互作用,表明SVP的K域(SVP第96～173位氨基酸区域)与FLC的K域(FLC第114～167位氨基酸区域)能够异源结合,是介导SVP与FLC蛋白互作的关键结构域。
Five SVP truncations (SVP1 ~ 5) and five FLC truncations (FLC1 ~ 5) were subcloned from the yeast recombinant plasmids pGADT7SVP and pGBKT7FLC to elucidate the interaction between SVP and FLC, ). The domains of SVP1 ~ 5 and FLC1 ~ 5 encoded proteins were MI, MIK, K, IKC and KC, respectively. Yeast two-hybrid system was used to construct yeast prey plasmid pGADT7SVP1 ~ 5 and bait plasmid pGBKT7FLC1 ~ 5, respectively, and transformed into yeast Y187 and Y2HGold. The yeast transformant Y187 [pGADT7SVP2-5] was able to fuse with Y2HGold [pGBKT7FLC] and grow blue colonies on the selective solid medium QDO / X / A, indicating that FLC can heterosexual to the truncated protein SVP2-5 In combination, the K domain of SVP (SVP3) acts independently on FLC proteins. In addition, Y187 [pGADT7SVP] × Y2HGold [pGBKT7FLC2-5] also activated the reporter genes AUR1-C, HIS3, ADE2 and MEL1 at the same time, indicating that FLC K domain (FLC3) can also act independently on SVP. Further study found that: Y187 [pGADT7SVP3] × Y2HGold [pGBKT7FLC3] forward hybridization and Y187 [pGADT7FLC3] × Y2HGold [pGBKT7SVP3] carrier exchange after hybridization can interact, indicating that the SVK K domain (SVP amino acid residues 96 to 173 ) Is able to heterodimerize with the K domain of FLC (amino acid residues 114-167 of FLC) and is a key domain that mediates SVP interaction with FLC protein.