目的 基于三七根转录组数据库，克隆三七皂苷合成关键酶细胞色素P450（cytochrome P450，CYP450）基因，对其进行生物信息学分析，明确其对丛枝菌根真菌（arbuscular mycorrhizal fungi，AMF）诱导的响应，为进一步研究其催化特性奠定基础。方法 基因PnCyp450_3开放阅读框（open reading frame，ORF）克隆及克隆载体构建依据相应试剂盒说明书，通过化学转化法及氨苄青霉素抗性标记筛选阳性克隆。利用各种在线工具或软件完成生物信息学分析及系统发育树构建；基因表达特性分析采用实时荧光定量（real-time fluorescence quantitative，qPCR）技术。结果 成功克隆了1个三七细胞色素P450超家族基因PnCyp450_3，其ORF全长1617 bp，编码538个氨基酸，预测蛋白相对分子质量为59 690，等电点（pI）为8.50，含有5个保守结构域。组织表达特性结果显示，在主根、须根部位，PnCyp450_3均可响应根内球囊霉（Glomus intraradices）的诱导而显著上调表达（P<0.05），其中主根相对表达量最高（1.85倍），须根次之（1.22倍）。结论 PnCyp450_3是一个编码538个氨基酸的CYP450超家族基因，在主根、须根中均可响应根内球囊霉的诱导而显著上调表达，为深入研究三七皂苷合成关键酶基因响应生物因子诱导的调控机制提供了数据支持，具有重要的参考价值。

Objective Based on the Panax notoginseng root transcriptome database, the key enzyme cytochrome P450 (CYP450) gene of P. notoginseng saponin synthesis was cloned and bioinformatically analyzed to clarify its response to the induction of arbuscular mycorrhizal fungi (AMF) and to lay a foundation for the further study on catalytic properties. Methods The clone of PnCyp450_3 ORF and the construction of cloning vector were carried out according to the corresponding kit instruction. The positive clone was gained by chemical conversion and ampicillin screening. Bioinformatics analysis and phylogenetic tree were generated by a series of online tools or software, while gene expression characteristics were analyzed by real-time fluorescence quantitative (qPCR). Results A P. notoginseng cytochrome P450 superfamily gene, PnCyp450_3, was successfully cloned with the open reading frame (ORF) of 1617 bp, encoding 538 amino acids. Predicted molecular weight and isoelectric point (pI) was 59 690 and 8.50, respectively. The results of tissue expression characteristics showed that the expression level of PnCyp450_3 was significantly up-regulated by Glomus intraradices (P < 0.05) in the rhizomes and fibrous roots with relative expression levels of 1.85 and 1.22. Conclusion PnCyp450_3, a CYP450 superfamily gene encoding 538 amino acids, was significantly up-regulated in both primary and fibrous roots in response to the induction of G. intraradices. This study provides data to support an in-depth study of the regulatory mechanism of biological factors to key enzyme-encoding genes on the notoginsenoside synthesis, which has important reference value.

R286.12

中央本级重大增减支项目“名贵中药资源可持续利用能力建设项目”（2060302-2101-24）；国家自然科学基金资助项目（82260743）；云南省科技厅应用基础计划面上项目（202201AT070219，202001AT070109，2019FB122）；云南省中医药应用基础研究联合专项（202001AZ070001-010，202101AZ070001-014）；云南省“万人计划”青年拔尖人才专项（YNWR-QNBJ-2020-279）；昆明市国际（对外）科技合作基地项目（GHJD-2021030）