大白菜渗透压应激活化蛋白激酶基因SRK2F的克隆与表达

以不耐盐、不耐旱的大白菜自交系SY-14-06为试材,提取根部总RNA,反转录为c DNA。根据大白菜SRK2F基因设计引物,PCR扩增SRK2F基因CDS序列1044bp。SRK2F编码347个氨基酸,预测分子量为39.3k D,理论等电点为4.88。利用p EASY-E1原核表达载体构建原核表达质粒p EASY-E1-SRK2F,转化表达菌株Transetta(DE3),通过SDS-PAGE检测该蛋白的表达。经Smart-embl预测其具有丝/苏氨酸激酶特有结构域,位于第4~260位氨基酸处。经Clustal X2比对,其与拟南芥同源性最高。最后利用镍离子金属螯合亲和层析介质对该蛋白进行纯化,得到了纯化的融合蛋白。

Cloning and Expression of SRK2F Gene in Chinese Cabbage

Taking non salt and drought tolerant inbred lines of Chinese Cabbage〔Brassica campestris L.spp. pekinensis（Lour）Olsson〕SY-14-06 as material，extracted total RNA from root，and reverse transcribed cDNA．According to SRK2F gene sequence of Brassica rapa，primers were designed and 1 044 bp open reading frame（ORF）was cloned．The SRK2F protein contained 347 aminoacids，with a prediction molecular weight of 39.3 kD and PI of 4.88．We construct the procayotic expressive plasmids pEASY-E1-SRK2F using pEASY-E1 vector．After transformation to Transetta（DE3），the expression of recombinant proteins were detected with SDSPAGE．The structural analysis of SRK2F though Smart-embl showed that it contained silk threonine structural domain，which was located at the position of 4-260 aminoacid residues．The ClustalX2 comparison indicated that the SRK2F had close genetic relationship with Arabidopsis thaliana．Finally，purified this protein by affinity chromatography medium，which was known as Nickel ion metal affinity chromatography medium．Thus，gained the purified fusion protein.