芥蓝1-脱氧-D-木酮糖-5-磷酸合成酶基因BaDXS1的克隆及原核表达

本研究以芥蓝为实验材料,采用同源克隆的方法分离出BaDXS1基因,其开放阅读框为2 139 bp,编码712个氨基酸。理化性质分析表明,BaDXS1蛋白的分子量为77.21 ku,等电点pI为8.59,不含跨膜区域,位于植物细胞叶绿体内。氨基酸序列比对发现,芥蓝DXS1与甘蓝、拟南芥、烟草、番茄等植物的DXS蛋白序列的一致性达到79%以上;系统进化树分析显示,芥蓝DXS1与甘蓝DXS的亲缘关系最近。构建原核表达载体pEASY-Blunt E1-BaDXS1,并对其进行诱导表达,发现该蛋白在大肠埃希菌体内主要以包涵体的形式存在。

Cloning and prokaryotic expression of 1-deoxy-D-xylulose-5-phosphate synthase encoding gene (BaDXS1) in Brassica alboglabra

The 1-deoxy-D-xylulose-5-phosphate synthase (DXS) encoding gene BaDXS1 was isolated from Chinese kale (Brassica alboglabra) leaves by using homologous cloning method. BaDXS1 contained an open reading frame of 2 139 bp in length, encoding 712 amino acids. Physical and chemical properties analysis indicated that the molecular weight of BaDXS1 protein is 77.21 ku, and the isoelectric point pI is 8.59. There was no transmembrane region, and BaDXS1 is located in chloroplast of plant cell. Amino acid sequences comparison results showed that the identities of BaDXS1 and those in Brassica oleracea, Arabidopsis thaliana, tobacco, tomato were all over 79%. Phylogenetic tree analysis showed that it had close relation with BoDXS. The prokaryotic expression vector pEASY-Blunt E1-BaDXS1 was constructed, and the protein was successfully expressed in the form of inclusion bodies in Escherichia coli.