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为深入研究芥菜开花信号整合子的两个核心调节因子SHORT VEGETATIVE PHASE(SVP)与FLOWERING LOCUS C(FLC)相互作用的分子机理,通过PCR扩增,从芥菜材料‘QJ’中分别克隆含EcoRⅠ/BamHⅠ双酶切位点的SVP和FLC编码区全长,并利用酵母双杂交体系,将FLC与GAL4报告基因DNA激活域融合(pGADT7FLC),SVP与GAL4报告基因DNA结合域融合(pGBKT7SVP)。两种重组质粒分别转化酵母Y187和Y2HGold后未出现自激活和毒性现象。融合的二倍体酵母(pGADT7FLC×pGBKT7SVP)能在选择性固体培养基QDO/X/A(SD/-Ade/-His/-Leu/-Trp/X-α-Gal/AbA)上生长,并且菌落呈蓝色。将诱饵质粒(pGBKT7SVP)与猎物质粒(pGADT7FLC)载体互换(pGADT7SVP、pGBKT7FLC),再次转化酵母后仍能融合成二倍体酵母(pGADT7SVP×pGBKT7FLC),并同时激活报告基因AUR1-C、HIS3、ADE2、MEL1,由此表明SVP与FLC蛋白能够相互结合。
In order to further study the molecular mechanism of the interaction between SHORT VEGETATIVE PHASE (SVP) and FLOWERING LOCUS C (FLC), we studied the molecular mechanism of interaction between flowering locus (FL) and FLOWERING LOCUS C (FLC) BamH I double restriction enzyme sites and the full-length of FLC coding region. Fusion of FLC with GAL4 reporter DNA activating domain (pGADT7FLC) and SVP with GAL4 reporter gene DNA binding domain (pGBKT7SVP) were performed by yeast two-hybrid system. The two recombinant plasmids transformed into yeast Y187 and Y2HGold did not show self-activation and toxicity. The fused diploid yeast (pGADT7FLC × pGBKT7SVP) was able to grow on selective solid medium QDO / X / A (SD / -Ade / -His / -Leu / -Trp / X-α-Gal / AbA) Colonies are blue. The bait plasmids (pGBKT7SVP) and prey plasmid (pGADT7FLC) vector were interchanged (pGADT7SVP, pGBKT7FLC), and then transformed into yeast and then transformed into diploid yeast (pGADT7SVP × pGBKT7FLC). At the same time, the reporter gene AUR1- ADE2, MEL1, indicating that SVP and FLC protein can bind to each other.