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目的从滇龙胆Gentiana rigescens幼叶中克隆单萜化合物合成的关键酶牻牛儿基焦磷酸合成酶基因GrGPPS,进行序列特征分析和原核表达。方法根据三年生滇龙胆转录组GrGPPS基因序列,设计特异性引物,通过RT-PCR扩增得到GrGPPS cDNA序列,并进行TA克隆、测序及序列分析;构建原核表达载体pGEX-4T-1-GrGPPS,转入Escherichia coli Rosetta(DE3)中,在37℃、1.0 mmol/L IPTG诱导下进行表达。结果 GrGPPS cDNA全长1 107 bp,编码369个氨基酸;序列分析表明,GrGPPS基因是异戊烯基合成酶家族的成员;氨基酸序列系统发育分析表明,GrGPPS与金鱼草AmGPPS亲缘关系最近;构建pGEX-4T-1-GrGPPS重组质粒,获得稳定的pGEX-4T-1-GrGPPS原核表达体系。SDS-PAGE结果表明所表达蛋白与预期蛋白大小一致。结论克隆了GrGPPS基因,建立pGEX-4T-1-GrGPPS稳定的原核表达体系,为进一步纯化和鉴定GPPS蛋白并研究其结构和功能奠定基础。
Objective To clone the gene GeGGPPS, a key enzyme in the synthesis of monoterpene compounds, from young leaves of gentiana rigescens, and analyze the characteristics of the gene and prokaryotic expression. Methods Based on the GrGPPS gene sequence of the three-year-old Yunnan gentian transcriptome, specific primers were designed and the GrGPPS cDNA sequence was amplified by RT-PCR. The GrGPPS cDNA was cloned, sequenced and sequenced. The prokaryotic expression vector pGEX-4T-1-GrGPPS , Transformed into Escherichia coli Rosetta (DE3) and expressed under induction of 1.0 mmol / L IPTG at 37 ° C. Results The GrGPPS cDNA was 1 107 bp in length and encoded 369 amino acids. The sequence analysis showed that the GrGPPS gene was a member of isopentenyl synthase family. Phylogenetic analysis showed that GrGPPS was closest to AmGPPS, 4T-1-GrGPPS recombinant plasmid to obtain a stable prokaryotic expression system pGEX-4T-1-GrGPPS. SDS-PAGE results showed that the expressed protein and expected protein size. Conclusion The GrGPPS gene was cloned and a stable prokaryotic expression system of pGEX-4T-1-GrGPPS was established, which laid the foundation of further purification and identification of GPPS protein and its structure and function.