目的 克隆三七总皂苷甲羟戊酸合成途径中的1个关键酶3-羟基-3-甲基戊二酸单酰辅酶A还原酶（3-hydroxy-3-methylglutaryl coenzyme-A reductase，HMGR）基因，为进一步研究HMGR蛋白的功能及开展三七皂苷的合成生物学研究奠定基础。方法 根据NCBI上已公布的同属人参的HMGR基因全长序列（登录号GU565097.1）设计特异性引物。利用RT-PCR技术，以三七愈伤组织总RNA反转录的cDNA为模板扩增基因片段；并对其编码的蛋白进行生物信息学预测和基因表达分析。结果 序列分析表明，获得的三七HMGR（命名为PnHMGR）基因的cDNA序列大小为1 893 bp，该序列与人参、刺五加和杜仲中HMGR基因的一致性分别达到98%、93%、80%，且含有大小为1 725 bp的开放阅读框，经Genbank查询为一新的cDNA。生物信息学预测PnHMGR基因编码蛋白包含2个跨膜区，不含信号肽，具有HMGR催化作用的活性中心结构域。实时荧光定量PCR检测到PnHMGR基因在三七细胞生长30 d表达量最高。结论 首次从药用植物三七中克隆得到HMGR基因的编码区序列，为进一步鉴定PnHMGR的功能和开展三七皂苷的合成生物学研究奠定了基础。

Objective Panax notoginseng is an important medicinal plant and its secondary metabolites, P. notoginseng saponins (PNS), synthesized by the mevalonate pathway are the active ingredients. The study on the gene of the key enzyme 3-hydroxy-3- methylglutaryl-coenzyme-A reductases (HMGR) in the mevalonate pathway is helpful for the regulation of PNS syntheses. Methods The primers were designed according to P. ginseng HMGR (accession number: GU565097.1) from NCBI. Total RNA was extracted from the callus of P. notoginseng. The fragment of HMGR gene was amplified by reverse transcription PCR technology and analyzed. Results Sequence analysis showed that the cDNA sequence of obtained fragment (PnHMGR) was 1 893 bp, containing an ORF spaning 1 725 bp, and exhibited 98%, 93%, and 80% sequence identity with HMGR in P. ginseng, Eleutherococcus senticosus, and Eucommia ulmoides. The cDNA was a new one as searching in the Genbank. The bioinformatic analysis showed that PnHMGR-encoding protein contained two transmembrane regions and the HMGR catalytic domain, without signal peptide. The expression level of PnHMGR was the highest when the callus of P. notoginseng has grown for 30 d. Conclusion It is the first time to report HMGR gene isolated from P. notoginseng. The results will provide a groundwork for exploring the molecular function of PnHMGR involved in PNS biosynthesis based on the synthetic biology of P. notoginseng.

国家自然科学基金资助项目（31260070，31060044）