目的 筛选茉莉酸甲酯（methyl jasmonate，MeJA）诱导下积雪草Centella asiatica中与三萜皂苷积累密切相关的关键结构基因，并阐明其表达特征。方法 采用200 μmol/L MeJA处理积雪草植株，利用高效液相色谱法测定不同时间点叶片中积雪草苷与羟基积雪草苷含量变化，并结合转录组数据开展加权基因共表达网络分析，筛选与皂苷积累相关的关键基因模块，进一步通过Mfuzz时序聚类划分基因亚簇，并经实时荧光定量PCR验证候选基因表达模式。结果 MeJA处理显著促进积雪草苷与羟基积雪草苷积累，含量分别提高18.28%和14.58%。加权基因共表达网络分析（weighted gene co-expression network analysis，WGCNA）共识别33个共表达模块，其中darkred模块与积雪草苷含量呈极显著正相关（P＜0.01）。该模块中286个基因经Mfuzz聚类划分为4个亚簇，其中Cluster 1～3共239个基因显著富集于萜类骨架、倍半萜及三萜生物合成途径。从中进一步鉴定出28个与积雪草苷合成直接相关的结构基因，其表达模式与皂苷积累趋势一致。qRT-PCR验证确认11个参与三萜骨架合成与修饰的关键基因在MeJA诱导后均显著上调，与转录组结果一致。结论 MeJA通过协同调控darkred模块中11个关键结构基因的表达，促进积雪草三萜皂苷生物合成。从多组学整合与实验验证角度揭示了MeJA对积雪草三萜皂苷积累的影响及相关基因表达特征，为后续功能解析与品种改良提供基因资源与理论依据。

Objective This study investigated the regulatory mechanism by which methyl jasmonate (MeJA) modulates triterpenoid saponin biosynthesis in Jixuecao (Centella asiatica). Methods Plants were treated with 200 μmol/L MeJA, and temporal changes in leaf asiaticoside and madecassoside contents were quantified by high-performance liquid chromatography (HPLC). Transcriptome data were analyzed using weighted gene co-expression network analysis (WGCNA) to identify modules significantly correlated with asiaticoside accumulation, followed by Mfuzz clustering to refine gene subclusters. Expression patterns of candidate key genes were validated by quantitative real-time PCR (qRT-PCR). Results MeJA treatment markedly promoted asiaticoside and madecassoside accumulation, increasing their contents by 18.28% and 14.58%, respectively. WGCNA revealed 33 co-expression modules, with the darkred module displaying a highly significant positive correlation with asiaticoside content (P < 0.01). Mfuzz clustering subdivided the 286 genes in this module into four subclusters; Notably, 239 genes in Clusters 1-3 were significantly enriched in the terpenoid backbone biosynthesis, sesquiterpenoid biosynthesis, and triterpenoid biosynthesis pathways. Twenty-eight structural genes directly involved in asiaticoside synthesis were identified, exhibiting expression profiles closely aligned with saponin accumulation. qRT-PCR confirmed significant MeJA-induced upregulation of 11 key structural genes associated with triterpenoid backbone synthesis and modification, consistent with the transcriptome findings. Conclusion MeJA enhances triterpenoid saponin biosynthesis in C. asiatica through coordinated upregulation of these 11 key structural genes within the darkred module. By integrating multi-omics profiling with experimental validation, this work systematically elucidates the molecular mechanisms underlying MeJA-induced triterpenoid saponin accumulation, offering valuable candidate genes for future functional characterization and genetic enhancement of C. asiatica.

R282.12

国家自然科学基金青年科学基金项目（32500333）；湖北省中医药管理局中医药创新团队项目（ZY2025J002）；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）