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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-site receptor kinase (MLPK) is a key positive regulator of self-incompatibility in cabbage, and the molecular mechanism of its involvement in self-incompatibility is unclear. In order to isolate the protein interacting with MLPK, MLPK kinase domain coding sequence (MLPKK) was amplified by PCR based on the analysis of MLPK domain. Two MLPK inactivating mutants were constructed by site-directed mutagenesis in vitro (Denoted as mlpk1 and mlpk2). Then, the prokaryotic expression plasmids p ET43.1a-MLPKK, p ET43.1a-mlpk1 and p ET43.1a-mlpk2 were constructed with p ET43.1a as a vector, and prokaryotic expressed and purified. The purified fusion proteins p ET43.1a-MLPKK, p ET43.1a-mlpk1 and p ET43.1a-mlpk2 were incubated with the total self-incompatibility Brassica oleracea ’A4’ total protein extract. After incubation, the fusion protein sequences 6 × His tag with Ni + binding characteristics, to capture the interaction protein MLPK, the establishment of a new method of separation of MLPK interacting proteins. Incubation of the products by SDS-PAGE electrophoresis showed that compared with the two mutant protein lanes, a candidate protein band interacting with MLPK was successfully obtained in the lane where the product was incubated with p ET43.1a-MLPKK and stigma total protein extract, This provides technical support for the subsequent identification of protein interaction and functional analysis.