目的 从滇龙胆幼叶中克隆萜类合成相关酶基因GrCYP450-17，进行序列分析和原核表达。方法 根据滇龙胆转录组中GrCYP450-17基因序列，设计引物，通过RT-PCR扩增得到GrCYP450-17 开放阅读框（ORF）序列，并进行TA克隆、测序和序列分析；构建原核表达载体pGEX-4T-1-GrCYP450-17，转入大肠杆菌Rosetta（DE3）中，在异丙基-β-D-硫代半乳糖苷（IPTG）诱导下进行表达。结果 GrCYP450-17 ORF全长1 545 bp，编码514个氨基酸。序列分析表明，GrCYP450-17基因是CYP714家族成员；蛋白质序列系统发育分析表明，GrCYP450-17与番茄SlCYP450亲缘关系最近。构建pGEX-4T-1-GrCYP450-17重组质粒，获得稳定的原核表达体系。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GrCYP450-17基因，建立其稳定的原核表达体系，为进一步纯化GrCYP450-17蛋白、研究其结构和功能奠定基础。

Objective To obtain the key enzyme gene GrCYP450 involving in the terpenoid biosynthesis, a CYP450-17 gene was cloned from young leaves of Gentiana rigescens, and its sequence analysis and prokaryotic expression were performed. Methods According to the GrCYP450-17 gene sequence of transcriptome of triennial G. rigescens, a pair of primers were designed, and the ORF (Open reading frame)of cDNA sequences was obtained by RT-PCR. Then TA cloning, sequencing, and sequence analysis were performed. Prokaryotic expression vector pGEX-4T-1-GrCYP450-17 was constructed and transformed into E. coli Rosetta (DE3) for the expression under the induction of Isopropyl β-D-1-Thiogalactopyranoside (IPTG.). Results The ORF of GrCYP450-17 has a length of 1 545 bp coding for 514 amino acids. Sequence analysis showed that GrCYP450-17 was the member of CYP714 family. Results of phylogenic analysis showed that GrCYP450-17 was close to SlCYP450 of tomato. The SDS-PAGE results showed that the expressed proteins were consistent with the anticipated size. Conclusion The GrCYP450-17 gene is successfully cloned, and the stable prokaryotic expression system is established. This study will provide a foundation for the further purification, structural and functional researches of GrCYP450-17 protein.

国家自然科学基金资助项目（81260608）；云南省教育厅科学研究基金重点项目（2013Z075）；科技部“十二五”国家科技支撑计划项目（2011BAI13B02-04）