甘蓝自交不亲和信号传导元件ARC1的体外表达及其与SRK相互作用验证

在甘蓝自交不亲和信号传导中ARC1和上游因子SRK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ARC1的编码序列, 构建ARC1原核表达质粒pET43.1a-ARC1，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-ARC1融合蛋白序列中的6×His标签与Ni⁺结合的特点建立了体外检测蛋白质相互作用的新方法, 并用该方法对ARC1与SRK的相互作用进行了检测。结果表明，在体外ARC1能与SRK相互作用并形成复合体，这为深入分析ARC1与SRK相互作用机理以及探讨ARC1与下游传导元件的相互作用提供了理论和技术基础。

Expression of ARCl in Vitro and Test of Interaction between ARC1 and SRK from Brassica oleracea L.in Signal Transduction Pathway of Self-Incompati-bility

Self-incompatibility (SI) is a widespread mechanism in flowering plants that prevents inbreeding and promotes outcrossing. Self-pollen recognition relies on the products of genes located at the S (self-incompatibility) locus. Significant progress has been made in understanding molecular interactions allowing stigmatic cells to recognize and reject self-pollen in Brassica during the past decades. Thus, the male and female determinants responsible for the self-incompatibility (SI) response have been identified. The structural features of these molecules strongly suggest that SI response is triggered by a ligand-receptor interaction. ARC1 is anArm Repeat Containing and also a downstream gene of SRK. ARC1 interacts with SRK probably in signal transduction pathway of self-incompatibility. In this study, with an aim to testify the interaction, the coding sequences of ARC1 were amplified from stigma mRNA of Brassica oleracea L., and inserted into the expression vector pET43.1a to construct the recombinant plasma pET43.1a-ARC1, transformed E. coli(BL21) and detected expression of the recombinant protein via SDS-PAGE. Using Co-Immunoprecipitation theory and characteristic of 6×His Tag combined with Ni+ in pET43.1a-ARC1, a new method was put forward for testing the interaction between proteins. Through the method the interaction between ARC1 and SRKwas analyzed, showing that ARC1 and SRK could act with each other to combine and form a complex. This research provides a theoretical and technical bases for further analyzing the mechanism of interaction between ARC1 and SRK, for probing into interaction between ARC1 and downstream targets.