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为了深入研究调控芥菜开花信号整合子的两个核心转录因子Flowering Locus C(FLC)和SHOTR VEGETATIVE PHASE(SVP)的作用机理和进行人工调控,以芥菜‘QJ’为材料,采用RT-PCR技术分别扩增FLC和SVP的编码序列,构建原核表达质粒pET43.1a-FLC、pET43.1a-SVP,转化宿主菌大肠杆菌BL21,通过SDS-PAGE检测该蛋白的表达。利用免疫共沉淀原理及pET43.1a-FLC、pET43.1a-SVP融合蛋白序列中的6×His标签能与Ni+结合的特点,对芥菜FLC与SVP的相互作用进行SDS-PAGE检测。结果表明:FLC与SVP能在体外相互作用并形成复合体,为深入分析FLC与SVP间的作用机理以及探讨其与下游开花信号整合子元件的相互作用提供了理论和技术基础。
In order to further study the mechanism and regulation of two core transcription factors Flowering Locus C (FLC) and SHOTR VEGETATIVE PHASE (SVP) which regulates the flowering signal integrin of Brassica juncea, we used mustard (QJ) as material and RT-PCR The coding sequences of FLC and SVP were amplified. The prokaryotic expression plasmids pET43.1a-FLC and pET43.1a-SVP were constructed and transformed into E. coli BL21. The expression of the protein was detected by SDS-PAGE. The interaction between FLC and SVP in Brassica juncea was detected by SDS-PAGE using the principle of co-immunoprecipitation and binding of 6 × His tag in pET43.1a-FLC and pET43.1a-SVP fusion proteins to Ni +. The results showed that FLC and SVP could interact and form complexes in vitro, which provided the theoretical and technical basis for further analysis of the interaction mechanism between FLC and SVP and the interaction with downstream flowering signal integron.