羽衣甘蓝BoMAPK4原核表达、抗体制备及蛋白表达分析

Prokaryotic Expression, Preparation Polyclonal Antibody and Protein Expression of BoMAPK4 of Ornamental Kale(Brassica oleracea var. acephala)

以羽衣甘蓝(Brassica oleracea var.acephala)自交不亲和系(S_(13-b)S_(13-b))为材料,提取柱头RNA,利用RT-PCR分离羽衣甘蓝柱头丝裂原活化蛋白激酶4(MAPK4)基因。结果表明:获得的Bo MAPK4 c DNA含有一个1 122 bp开放阅读框,编码373个氨基酸,具有丝氨酸/苏氨酸结构域,无信号肽和跨膜结构。羽衣甘蓝Bo MAPK4与油菜(B.napus)Bn MAPK4、芜菁(B.rapa)Br MAPK4、拟南芥(Arabidopsis thaliana)At MAPK4的氨基酸序列一致性分别为99.7%、99.5%、95.4%。将Bo MAPK4编码区的序列构建到原核表达载体p ET-14b上,并转化到BL21(DE3)大肠杆菌(Escherichia coli)中进行原核表达。SDS-PAGE结果显示,在分子量大小约45 k Da处有Bo MAPK4重组蛋白特异性表达。利用亲和层析的方法获得高纯度的重组Bo MAPK4蛋白。利用获得的重组Bo MAPK4蛋白免疫小鼠,制备Bo MAPK4的多克隆抗体。提取羽衣甘蓝萼片、花瓣、花药、柱头、花柱和子房的总蛋白,利用免疫印迹的方法进行蛋白表达检测,结果发现Bo MAPK4在花瓣、花药和子房中表达的较少,在萼片和花柱中表达的较多,在柱头中的表达量最高。这些研究为探讨Bo MAPK4的生物学功能奠定了基础。

The mitogen-activated protein kinase 4(MAPK4)c DNA were isolated from the stigma of ornamental kale(Brassica oleracea var.acephala)self-incompatibility line(S_(13-b)S_(13-b))by RT-PCR.The obtained Bo MAPK4c DNA contained a 1 122 bp open reading frame in length,encoded 373 amino acids,and had a serine/threonine domain,no signal peptide and transmembrane structure.The deduced amino acid sequence of Bo MAPK4 shared 99.7%,99.5%and 95.4%identity with Brassica napus Bn MAPK4,Brassica rapa Br MAPK4,Arabidopsis thaliana At MAPK4,respectively.The ORF of Bo MAPK4 was constructed into the prokaryotic expression vector p ET-14b and transformed into Escherichia coli BL21(DE3)for expression.SDS-PAGE showed that Bo MAPK4 protein was specifically expressed at a molecular weight of 45 k Da.The recombinant Bo MAPK4 was obtained by affinity chromatography,and the polyclonal antibody against Bo MAPK4 was prepared by immunizing mice.The total proteins of sepals,petals,anthers,stigmas,styles and ovaries of ornamental kale were extracted,and protein expression was detected by Western blot.The results showed that Bo MAPK4 was less expressed in petals,anthers and ovary,more expressed in sepals and styles,and the highest expressed in stigmas.The study lays the foundation for exploring the biological functions of Bo MAPK4.