关键词: 甘蓝, 自交不亲和, M&ndash, 位点受体激酶（MLPK）, 互作蛋白, 分离方法

Abstract:

M-locus receptor kinase（MLPK）is a positive regulatory element for Brassica self- incompatibility（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 mRNA 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 pET43.1a to construct the recombinant plasmid pET43.1a-MLPKK，pET43.1a-mlpk1 and pET43.1a-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 pET43.1a-MLPKK，pET43.1a-mlpk1 and pET43.1a-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 pET43.1a-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.

Key words: Brassica oleracea, self-incompatibility, M-locus receptor kinase, interacting protein, separation method