利用酵母双杂交法检测甘蓝SCR与SRK之间的相互作用

摘要

为深入研究甘蓝S位点受体激酶(SRK)和S位点富含半胱氨酸蛋白(SCR)间相互识别的分子机理,以具有典型自交不亲和性的甘蓝D3为材料,利用巢式PCR分别扩增SRK胞外域(eSRK)和SCR,通过酵母双杂交检测二者之间的相互作用,并利用同源重组技术将eSRK与GAL4报告基因DNA活化域融合(pGADT7eSRK)以及SCR与GAL4报告基因DNA结合域融合(pGBKT7SCR).两种重组质粒分别转化酵母Y187和Y2HGold后未出现自激活现象.融合的二倍体酵母(pGADT7eSRK×pGBKT7SCR)能在选择性固体培养基(SD/-Ade/-His/-Leu/-Trp/X-a-Gal/AbA)上生长,并且菌落呈蓝色.结果表明,eSRK与SCR蛋门能够相互结合,为深入研究二者作用位点成功建立了一个技术平台.%For further study on mechanism of the mutual recognition between S-locus receptor kinase (SRK) and S-locus Cysteine-rich protein (SCR) in Brassica, the extracellular domains of SRK (named as eSRK) and SCR were amplified by nested PCR using Brassica oleracea L.“D3” with the trait of typical self-incompatibility (SI), and the interactions between eSRK and SCR was detected by the yeast two-hybrid system.Then the full-length eSRK and the SCR were fused to the Gal4 DNA activation domain (designated pGADT7eSRK) and the Gal4 DNA binding domain (designated pGBKT7SCR) by the gene homologous recombination technique respectively.Then the pGADT7eSRK plasmid and the pGBKT7SCR were transformed into the Y187 yeast strain and the Y2HGold yeast strain respectively.The two transformed yeast strains did not exhibit autoactivation.The diploids, which were fused by the transformed Y2HGold yeast strain and the transformed Y187 yeast strain, grew on selective agar plates (SD/–Ade/–His/– Leu/–Trp/X-a-Gal/AbA), and the resulting colonies were blue, which strongly indicated that eSRK and SCR could combine with each other.