目的 旨在克隆获得远志Polygala tenuifolia 细胞色素P450（cytochrome P450，CYP450）基因CYP72A546的基因开放阅读框（open reading frame，ORF），并对其理化特性、系统进化关系、组织表达、激素与非生物胁迫下的表达等展开分析。方法 基于远志全长转录组文库筛选并克隆远志细胞色素P450家族基因PtCYP72A546，通过生物信息学在线软件分析其编码的氨基酸序列，预测蛋白理化性质、结构域等分子特征；利用MEGA11分析进化关系，并采用实时荧光定量PCR（qPCR）方法检测不同组织、激素及非生物胁迫的相对表达量。结果 从远志中成功克隆得到远志细胞色素P450家族基因PtCYP72A546，其ORF序列全长共计1 560 bp，编码519个氨基酸，相对分子质量为59 770，系疏水性稳定蛋白，含有1个保守结构域，不具有跨膜结构，二级结构主要为α-螺旋和无规卷曲，亚细胞定位分析表明PtCYP72A546-GFP融合蛋白定位于内质网。qPCR结果显示，远志根中CYP72A546表达量显著高于茎和叶片；200 μmol/L脱落酸（abscisic acid，ABA）和200 μmol/L壳聚糖（chitosan，CHT）处理下，CYP72A546均于12 h达峰值，分别为空白对照的7.45倍和5.49倍；在非生物（干旱、盐）胁迫处理下，CYP72A546的表达均先受抑制（6 h），再上调后缓慢下降。结论 通过对远志CYP72A546基因的克隆、鉴定与分析，为深入研究其在远志三萜皂苷合成途径的功能奠定基础。

Objective To clone the open reading frame (ORF) of cytochrome P450 (CYP450) gene CYP72A546 from Yuanzhi (Polygala tenuifolia Willd.), and analyze its physicochemical properties, phylogenetic relationship, tissue expression, expression under hormone and abiotic stress. Methods Based on the full-length transcriptome library of P. tenuifolia, the cytochrome P450 family gene PtCYP72A546 of P. tenuifolia was screened and cloned. The amino acid sequence encoded by it was analyzed by bioinformatics online software, and the molecular characteristics such as physical and chemical properties and structural domains of the protein were predicted. The evolutionary relationship was analyzed by MEGA11, and the relative expression levels of different tissues, hormones and abiotic stresses were detected by real-time fluorescence quantitative PCR (qPCR). Results The cytochrome P450 family gene PtCYP72A546 was successfully cloned from P. tenuifolia. The full-length ORF sequence was 1 560 bp, encoding 519 amino acids with a relative molecular mass of 59 770. It was a hydrophobic stable protein with a conserved domain and no transmembrane structure. The secondary structure was mainly α-helix and random coil. Subcellular localization analysis showed that the PtCYP72A546-GFP fusion protein was located in the endoplasmic reticulum. The results of qPCR showed that the expression of CYP72A546 in roots was significantly higher than that in stems and leaves. Under the treatment of 200 μmol/L abscisic acid (ABA) and 200 μmol/L chitosan (CTS), CYP72A546 reached the peak at 12 h, which was 7.45 times and 5.49 times that of CK, respectively. Under abiotic (PEG6000 and NaCl) stress treatments, the expression of CYP72A546 was first inhibited (6 h), then up-regulated and then slowly decreased. Conclusion The cloning, identification and analysis of the CYP72A546 gene in P. tenuifolia laid a foundation for further study on its function in the synthesis pathway of triterpenoid saponins in P. tenuifolia.

国家自然科学基金资助项目（82003899）；陕西省科技厅社发攻关一般项目（2021SF-364,2023-YBSF-036）；陕西中医药大学“秦药”品质评价及资源开发学科创新团队项目（2019-QN01）；陕西省教育厅一般专项（22JK0279）