甘蓝MLPK的原核表达及其互作蛋白分离体系的建立

Prokaryotic Expression of MLPK and the Establishment of Separation System of Its Interacting Proteins in Brassica oleracea

M–位点受体激酶(MLPK)是甘蓝自交不亲和反应的正向调控关键元件,其参与自交不亲和反应的分子机制尚不明确。为了分离与MLPK相互作用的蛋白,在分析了MLPK功能域的基础上采用PCR技术扩增了MLPK激酶结构域编码序列(记为MLPKK),通过体外定点突变技术构建了两种MLPK失活突变体(记为mlpk1和mlpk2),然后以p ET43.1a为载体构建了原核表达质粒p ET43.1a-MLPKK、p ET43.1a-mlpk1和p ET43.1a-mlpk2,并进行了原核表达和纯化。纯化的融合蛋白p ET43.1a-MLPKK、p ET43.1a-mlpk1和p ET43.1a-mlpk2分别与高度自交不亲和甘蓝‘A4’柱头总蛋白提取液进行孵育,孵育后利用融合蛋白序列中的6×His标签与Ni+结合的特性,钓取MLPK的互作蛋白,建立了分离MLPK互作蛋白的新方法。孵育产物经SDS-PAGE电泳显示,与两个突变体蛋白泳道对比,在p ET43.1a-MLPKK与柱头总蛋白提取液孵育产物的泳道中成功获得了候选的与MLPK互作的蛋白条带,这为后续互作蛋白质谱鉴定以及功能解析提供了技术支持。

M-locus receptor kinase（MLPK）is a positive regulatory element for Brassica selfincompatibility（SI）signaling,and MLPK＇s cellular mechanism in this response remains unknown. In this study,for the separation and identification of interacting proteins with MLPK during the course of SI,the coding sequences of MLPK kinase domain（named MLPKK）were amplified from stigma m RNA of Brassica oleracea,and two kinds of MLPK mutants（named mlpk1 and mlpk2）deficient in kinase activity were prepared by site-directed mutagenesis. Then,they were inserted into the expression vector p ET43.1a to construct the recombinant plasmid p ET43.1a-MLPKK,p ET43.1a-mlpk1 and p ET43.1a-mlpk2. The recombinant proteins were respectively expressed in E. coli（BL21）and purified. Using the characteristic of 6× His Tag of fusion protein combined with Ni＋,the purified fusion proteins p ET43.1a-MLPKK,p ET43.1a-mlpk1 and p ET43.1a-mlpk2 respectively were incubated with total protein extracts from stigmas of highly self-incompatible Brassica oleracea,fished interacting proteins with MLPK,and established a new method for separation of interacting proteins in vitro. The result of incubation products by SDS-PAGE electrophoresis showed that the lane of incubation products which p ET43.1a-MLPKK were incubated with total protein extracts successfully obtained candidate interacting proteins bands,compared with two mutant protein lanes. This research provides technical support for mass spectrum identification and functional explanation of interacting proteins.