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甘蓝抽薹开花抑制因子FLC与SVP可能存在相互作用,从而调节开花时间。为进一步的验证该相互作用,以甘蓝‘ZQ’为材料,扩增了594 bp的FLC cDNA和726 bp的SVP cDNA序列,它们分别编码197和241个氨基酸,且均属MIKC型蛋白。NCBI比对发现FLC与BoFLC3同源性高达98%,SVP与BoSVP-a同源性高达99%。将甘蓝FLC和SVP分别与原核表达载体pET43.1a融合,构建重组质粒pET43.1a-FLC、pET43.1a-SVP,并转化宿主菌大肠杆菌BL21,均能够在体外表达目的蛋白。利用免疫共沉淀的原理及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,结合SDSPAGE,检测到体外表达蛋白FLC与SVP能相互作用并形成复合体,这为深入研究FLC与SVP互作机理及探讨其与抽薹开花因子的相互作用提供了理论依据和技术基础。
Flowering time can be regulated by the interaction between flowering inhibition factor FLC and SVP in cabbage. To further verify this interaction, the 594-bp FLC cDNA and the 726-bp SVP cDNA sequence were amplified using the ’ZQ’ cabbage, which encodes 197 and 241 amino acids, respectively, and belongs to the MIKC type. NCBI alignment found that FLC and BoFLC3 homology of up to 98%, SVP and BoSVP-a homology of up to 99%. The recombinant plasmids pET43.1a-FLC and pET43.1a-SVP were fused with the prokaryotic expression vector pET43.1a respectively and transformed into E. coli BL21, which expressed the target protein in vitro. The immunoprecipitation method and the binding characteristics of 6 × His tag in pET43.1a-FLC and pET43.1a-SVP fusion protein with Ni + and the combination of SDSPAGE and the expression of FLC and SVP in vitro were detected. This provides a theoretical basis and a technical basis for further research on the interaction mechanism between FLC and SVP and the interaction between them and bolting and flowering factors.