【摘要】 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SRKE1)编码序列,构建了THL1原核表达质粒pET43.1-THL1,SRKE1原核表达质粒pET431-SRKE1和真核表达质粒pPIC9K-SRKE1,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SRKE1的相互作用进行了检测,结果表明THL1可与SRKE1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。更多还原

【Abstract】 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 SRKE1) 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-SRKE1, and pPIC9K-SRKE1. 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 SRKE1 and THL1 was analyzed, showing that SRKE1 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.更多还原