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为探明芥菜开花负调因子SVP、FLC自身聚合的分子机制及其蛋白作用模式,利用酵母双杂交体系,分别对SVP、FLC蛋白自身聚合及其作用强度进行研究。结果表明:酵母菌Y187转化子Y187-pGADT7SVP和Y187-pGADT7SVP2~5均能与酵母菌Y2HGold转化子Y2HGold-pGBKT7SVP融合,并可在选择性固体培养基QDO/X/A上长出蓝色菌落,而Y187-pGADT7SVP1×Y2HGold-pGBKT7SVP不能在QDO/X/A生长。说明SVP蛋白能自身聚合,且与截短体SVP2~5同源结合,SVP蛋白自身聚合需要核心作用域K域参与。尽管MI域不能单独介导SVP自身聚合,但它的存在却能使SVP自身聚合作用增强,C域有可能会削弱该作用。同时,Y2HGold-pGBKT7FLC和Y2HGold-pGBKT7FLC2~5也能与Y187-pGADT7FLC融合,同时激活报告基因AUR1-C、HIS3、ADE2、MEL1,FLC能与截短体FLC2~5同源互作。K域是FLC蛋白自身聚合必须的,I域会增强这一作用。SVP和FLC的核心作用域K域均由K1、K2和K3亚域组成,形成3个经典的α螺旋,K域有9个高度保守的氨基酸位点及蛋白互作的结构模体(亮氨酸拉链)。
In order to explore the molecular mechanisms of SVP and FLC self-polymerization and the mode of protein action, we studied the self-polymerization of SVP and FLC protein and the intensity of action by yeast two-hybrid system. The results showed that the yeast Y187 transformants Y187-pGADT7SVP and Y187-pGADT7SVP2-5 fused with the yeast Y2HGold transformant Y2HGold-pGBKT7SVP, and blue colonies could grow on the selective solid medium QDO / X / A, While Y187-pGADT7SVP1 × Y2HGold-pGBKT7SVP can not grow on QDO / X / A. It means that the SVP protein can polymerize by itself, and it is homologous to the truncated SVP2 ~ 5. The self-polymerization of SVP protein needs the core domain K domain to participate. Although the MI domain can not mediate SVP self-polymerization alone, its presence can enhance the self-polymerization of SVP, which may be weakened by C-domain. At the same time, Y2HGold-pGBKT7FLC and Y2HGold-pGBKT7FLC2 ~ 5 could also be fused with Y187-pGADT7FLC, and the homologues of AUR1-C, HIS3, ADE2, MEL1 and FLC could interact with the truncated FLC2-5. The K domain is necessary for the FLC protein to polymerize itself, and I domain enhances this effect. The core K domains of SVP and FLC are composed of K1, K2 and K3 subtypes, forming three classical α-helices. There are nine highly conserved amino acid sites in K domain and protein interacting structural motifs Acid zipper).