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开花是植物由营养生长向生殖生长的转换点,该过程受内源基因与环境信号共同调控。FLC(Flowering locus C)和SVP(Short vegetative phase)是开花途径中关键的抑制开花因子。本文利用酵母双杂交技术深入研究白桦开花抑制因子FLC与SVP的自身聚合作用的分子机理。从重组质粒p GBKT7-BpFLC、p GBKT7-Bp SVP分别克隆出6个BpFLC截短体[BpFLC1(aa1~73)、BpFLC2(aa1~173)、BpFLC3(aa74~173)、BpFLC4(aa60~208)、BpFLC5(aa74~208)和BpFLC6(aa174~208)]和6个BpSVP截短体[BpSVP1(aa1~68)、Bp SVP2(aa1~172)、Bp SVP3(aa69~172)、BpSVP4(aa60~225)、BpSVP5(aa69~225)和Bp SVP6(aa173~225)],分别编码MADS型蛋白的MI、MIK、K、IKC、KC和C域。在酵母Y2HGold菌中,分别共转质粒pGBKT7-BpFLC1~6×pGADT7-BpFLC及p GBKT7-BpSVP×pGADT7-BpSVP1~6。酵母转化子Y2HGold[p GBKT7-BpFLC×p GADT7-BpFLC]、Y2HGold[pGBKT7-BpFLC2~5×pGADT7-BpFLC],可在选择性固体培养基TDO(SD/-Leu/-Trp/-His)、QTO/A(SD/-Leu/-Trp/-His/-Ade/Ab A)上生长,并在QDO/A/X(SD/-Leu/-Trp/-His/-Ade/AbA/X-α-gal)上长出蓝色菌落,表明BpFLC能与自身及截短体蛋白BpFLC2~5同源结合。此外酵母Y2HGold[pGBKT7-BpSVP×pGADT7-Bp SVP]、Y2HGold[pGBKT7-Bp SVP×p GADT7-BpSVP2~5]也能同时激活报告基因AUR1-C、HIS3、ADE2、MEL1,说明BpSVP能与自身及截短体蛋白BpSVP2~5同源结合。对保守区的序列结构进一步分析发现:BpFLC与BpSVP的K域是它们能够同源结合,介导BpFLC与BpSVP自身形成同源二聚体的关键。
Flowering is the switch from vegetative growth to reproductive growth and is regulated by both endogenous genes and environmental signals. Flowering locus C (FLC) and Short vegetative phase (SVP) are key flowering-inhibiting flowering factors in the flowering pathway. In this paper, the yeast two-hybrid technique was used to study the molecular mechanism of the self-polymerization of flowering inhibitors FLC and SVP. Six BpFLC truncations (aa1-73, BpFLC2 (aa1-173), BpFLC3 (aa74-173), BpFLC4 (aa60-208)) were cloned from recombinant plasmids pGBKT7-BpFLC and pGBKT7- (Aa1 ~ 68), Bp SVP2 (aa1 ~ 172), Bp SVP3 (aa69 ~ 172), BpSVP4 (aa6 ~ 208) and BpFLC6 (aa174 ~ 208) 225, BpSVP5 (aa69-225) and Bp SVP6 (aa173-225)], encoding MI, MIK, K, IKC, KC and C domains of MADS type proteins, respectively. Plasmids pGBKT7-BpFLC1 ~ 6 × pGADT7-BpFLC and p GBKT7-BpSVP × pGADT7-BpSVP1 ~ 6 were co-transformed in Y2HGold yeast respectively. Yeast transformants Y2HGold [p GBKT7-BpFLC × p GADT7-BpFLC], Y2HGold [pGBKT7-BpFLC2-5 × pGADT7-BpFLC] can be cultured in selective solid medium TDO (SD / -Leu / -Trp / -His) QTO / A (SD / -Leu / -Trp / -His / -Ade / Ab A) and quantified on QDO / A / X (SD / -Leu / -Trp / -His / -Ade / AbA / X- α-gal), indicating that BpFLC can homologously bind to itself and the truncated protein BpFLC2-5. In addition, Y2HGold [pGBKT7-BpSVP × pGADT7-Bp SVP] and Y2HGold [pGBKT7-Bp SVP × pGADT7-BpSVP2-5] also activated the AUR1-C, HIS3, ADE2 and MEL1 reporter genes simultaneously. Truncated protein BpSVP2 ~ 5 homologous binding. Further analysis of the sequence structure of the conserved regions revealed that the K domains of BpFLC and BpSVP are the keys to their homologous binding and mediate the formation of homodimers between BpFLC and BpSVP itself.