甘蓝自交不亲和信号传导中THL1和SRK相互作用的体外检测

在甘蓝自交不亲和信号传导中SRK和THL1之间可能存在相互作用。为进一步证实该相互作用, 以甘蓝E1为材料, 采用RT-PCR技术扩增了THL1和SRK激酶结构域(记为SRK_E1)编码序列, 构建了THL1原核表达质粒pET43.1-THL1, SRK_E1原核表达质粒pET431-SRK_E1和真核表达质粒pPIC9K-SRK_E1, 分别在宿主菌大肠杆菌BL21和毕赤酵母GS115中进行表达。THL1融合蛋白经胰岛素检测, 表明表达的THL1有氧化还原活性。建立了体外检测蛋白质相互作用的新方法, 并用该方法对THL1与SRK_E1的相互作用进行了检测, 结果表明THL1可与SRK_E1相互作用并形成稳定的复合体, 这为深入分析THL1与SRK相互作用机理提供了理论和技术基础。

Test of Interaction between THL1 and SRK from Brassica oleracea L. in Self-Incompatibility Signaling Process

The SRK and THL1 from Brassica oleracea L. act probably with each other in self-incompatibility signal process. In present study, with an aim to testify the interaction, the coding sequences of THL1 and SRK kinase domain(named as SRK_E1) were amplified from stigma mRNA of Brassica oleracea L., and inserted into the expression vector pET43.1 and yeast expression vec-tor pPIC9K to construct the recombinant plasma pET43.1-THL1, pET43.1-SRK_E1, and pPIC9K-SRK_E1. The recombinant protein were expressed in E. coli(BL21) and Pichia pastoris (GS115), respectively. Thioredoxin activity was tested by the ability of THL1 reducing insulin, showing that THL1 had oxidation/reduction activity. A new method was put forward for testing the interaction between proteins, through the method the interaction between SRK_E1 and THL1 was analyzed, showing that SRK_E1 and THL1 could act with each other to combine and form a stable complex. This research provides a theoretical and technical bases for fur-ther analysis of the interaction mechanism between SRK and THL1.