目的 克隆积雪草Centella asiatica中参与三萜皂苷C-6β羟基化修饰的细胞色素P450基因（CaCYP2），系统解析其分子特征、组织表达模式及亚细胞定位，并实现原核可溶性表达，为阐明其在三萜生物合成中的功能提供实验基础。方法 从积雪草cDNA中克隆CaCYP2全长序列，并进行生物信息学分析，采用实时荧光定量PCR检测其组织特异性表达情况；利用无缝克隆技术构建酵母双杂交载体、原核表达载体及亚细胞定位载体，分别进行自激活检测、重组蛋白表达及烟草瞬时定位分析。结果 成功克隆CaCYP2基因，其开放阅读框（open reading frame，ORF）1 443 bp，编码480个氨基酸，属于CYP716家族，命名为CYP716E116。预测蛋白相对分子质量为54 425.09，理论等电点8.98，呈亲水性，含典型CYP90-like保守结构域及1个跨膜结构域。亚细胞定位结果显示，该蛋白定位于内质网。qRT-PCR分析表明，CaCYP2在叶片中表达量最高，是根中表达量的6.2倍（P＜0.000 1），与其他组织存在显著差异；原核表达结果表明，经异丙基硫代半乳糖苷（isopropylβ-D-thiogalactoside，IPTG）诱导后获得相对分子质量约为91 300的可溶性重组蛋白（含40 300的标签蛋白）；酵母双杂交实验证实，酵母双杂交载体无自激活活性，可用于后续互作蛋白筛选。结论 成功克隆CaCYP2基因，明确其分子结构特征、组织表达特异性及内质网定位，证实其可在原核系统中进行可溶性表达，为深入研究其催化功能及在积雪草三萜皂苷生物合成途径中的调控作用奠定重要的基础。

Objective To clone the cytochrome P450 gene (CaCYP2) involved in C-6β hydroxylation modification of triterpenoid saponins in Centella asiatica, systematically analyze its molecular characteristics, tissue expression pattern and subcellular localization, and achieve prokaryotic soluble expression, so as to provide experimental basis for clarifying its function in triterpenoid biosynthesis. Methods The full-length sequence of CaCYP2 was cloned from C. asiatica cDNA and subjected to bioinformatics analysis. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was performed to detect the gene specific expression in different tissues. Seamless cloning technology was employed to construct yeast two-hybrid vector, prokaryotic expression vector and subcellular localization vector for self-activation detection, recombinant protein expression and tobacco transient localization analysis, respectively. Results The CaCYP2 gene was successfully cloned. Its open reading frame (ORF) was 1 443 bp, encoding 480 amino acids, and it belonged to the CYP716 family, hence named CYP716E116. The predicted protein had a relative molecular mass of 54 430 and a theoretical isoelectric point of 8.98, showing hydrophilicity. It contained a typical CYP90-like conserved domain and one transmembrane domain. Subcellular localization results indicated that this protein was localized in the endoplasmic reticulum. qRT-PCR analysis showed that the expression level of CaCYP2 was highest in the leaves, which was 6.2 times higher than that in the roots (P < 0.000 1), showing significant differences compared to other tissues. Prokaryotic expression results indicated that a soluble recombinant protein with a relative molecular mass of approximately 91 300 (including a 40 300 tag protein) was obtained after isopropyl β-D-thiogalactoside (IPTG) induction. The yeast two-hybrid experiment confirmed that the yeast two-hybrid vector had no self-activating activity and could be used for subsequent screening of interacting proteins. Conclusion This study successfully cloned the CaCYP2 gene and characterized its molecular structure, tissue-specific expression pattern, and endoplasmic reticulum localization. It also confirmed that the protein can be expressed solubly in a prokaryotic system. These findings lay an important foundation for further investigation into its catalytic function and regulatory role in the biosynthetic pathway of triterpenoid saponins in C. asiatica.

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中央引导地方科技发展资金项目(桂科ZY24212031); 广西自然科学基金项目(2026GXNSFBA00640057,2023GXNSFAA026330); 广西岐黄学者培养项目(GXQH202402)