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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 To clone Ae. Albicans synthase (AeFPS) gene and perform prokaryotic expression. Methods Aralia elata was used as material, specific primers were designed and RT-PCR method was used to clone AeFPS gene. The gene was inserted into prokaryotic expression vector pET-28a cut with Sca I / BamH I and transformed into E.coli BL21. At 28 ℃, induced by IPTG for 5 h, SDS-PAGE electrophoresis analysis, detected significant differences in the band, Western blotting further detection of the target gene. Results The full-length cDNA of AeFPS was cloned into the cDNA library. The full-length cDNA of AeFPS was 1 040 bp in length and contained a 1029 bp open reading frame (ORF) encoding 342 amino acids. GenBank accession number was HM219226.1. The prokaryotic expression plasmid pET28a / AeFPS was successfully constructed. AeFPS protein was highly expressed in BL21, and the target protein was identified by SDS-PAGE and Western blotting. Conclusion The AeFPS gene was obtained for the first time and successfully expressed in prokaryotic vector, which laid the foundation for studying the activity and biochemical function of AeFPS protein.