龙牙楤木三萜皂苷合成途径关键酶AeFPS基因的克隆及原核表达

Cloning and prokaryotic expressing of AeFPS gene:a key enzyme of triterpene saponins biosynthesis in Aralia elata

目的克隆龙牙楤木法呢二磷酸合成酶(AeFPS)基因并进行原核表达。方法以龙牙楤木为材料,采用RT-PCR方法,设计特异引物,克隆AeFPS基因,并将该基因定向插入Sca I/BamH I切开的原核表达载体pET-28a上,转化大肠杆菌BL21,于28℃,IPTG诱导5 h,经SDS-PAGE电泳分析,检测出明显的差异条带,Western blotting进一步检测其为目的基因。结果克隆到AeFPS基因cDNA全长为1 040 bp,含有1 029 bp的开放阅读框(ORF),编码342个氨基酸,GenBank登录号为HM219226.1。成功构建了原核表达质粒pET28a/AeFPS、AeFPS蛋白在BL21中高度表达,SDS-PAGE和Western blotting鉴定了目标蛋白。结论首次获得了AeFPS基因,并将其连入原核载体中成功表达,为研究AeFPS蛋白的活性及生化功能奠定了基础。

Objective Cloning and prokaryotic expressing of farnesyl diphosphate synthase gene（abbreviated as AeFPS） in Aralia elata.Methods The AeFPS gene was amplified by RT-PCR with specific primers from A.elata and inserted into the prokaryotic expression vector pET-28a which is digested by Sca I and BamH I,then the recombinant vector was transformed into BL21 stain.After induced by IPTG at 28 ℃ for 5 h,the obvious difference between bands could be seen by SDS-PAGE and the AeFPS protein was detected by Western blotting too.Results The full-length cDNA of AeFPS（GenBank accession Number：HM219226.1） was 1 040 bp and contained a 1 029 bp open reading frame（ORF） encoding a polypeptide of 342 amino acids.The prokaryotic recombined plasmid pET28/AeFPS has been successfully constructed.The BL21 transformed recombined plasmid pET28/AeFPS had expressed AeFPS recombined protein effectively.The protein was detected by SDS-PAGE and Western blotting.Conclusion It is the first report that AeFPS gene could be cloned and expressed in Escherichia coli.This work is helpful for investigating the activities or other physiological functions of FPS protein.