摘要

长春花 (Catharanthus roseus) 作为一种多年生的药用植物，不仅在我国有着传统应用还因其能够合成多种具有显著药用价值的单萜吲哚生物碱 (Monoterpenoid Indole Alkaloids, MIA) ，尤其是抗癌药物长春碱和长春新碱而备受关注。为深入解析长春花中MIA的生物合成途径及其调控机制，本研究采用多组学策略，分析长春花液泡在MIA代谢中的作用并鉴定一具有异胡豆苷合成活性的蛋白STR2，同时筛选到长春碱合成相关的7个关键转录因子。

液泡作为植物细胞中的重要细胞器，不仅参与细胞的渗透调节、物质储存和降解，还在次生代谢产物的合成与积累中发挥关键作用。在长春花中，所有MIA的共同前体异胡豆苷(strictosidine)在液泡中被合成，这提示液泡在MIA生物合成途径中可能具有重要地位。为进一步探究液泡在该过程中的作用，本研究首先优化了长春花叶片细胞液泡的分离与纯化方法，通过结合渗透热激处理和密度梯度离心技术，成功获得了高质量的液泡样本。后利用液相色谱-质谱联用技术 (LC-MS/MS) 和超高效液相色谱-质谱联用技术 (UPLC-MS/MS) 对长春花液泡和原生质体样本进行了蛋白质组学和代谢组学测定分析。

蛋白质组学结果显示，液泡中富集了大量与物质代谢、转运及细胞解毒相关的蛋白质，这些蛋白质在维持液泡功能及MIA生物合成途径中发挥重要作用。另发现一些与离子转运、水解酶活性和转运蛋白功能相关的差异表达蛋白，这些蛋白可能参与了MIA前体的合成、转运和积累过程。代谢组学分析则进一步揭示了液泡中代谢物的组成，通过对比长春花叶片细胞液泡和原生质体的代谢物信息，发现液泡中积累了大量的生物碱、酚酸、脂质和黄酮类化合物，尤其是吲哚生物碱如文多灵和长春质碱等在液泡中的富集程度显著高于原生质体。这些发现不仅证实液泡作为MIA储存库的功能，还提示液泡在MIA生物合成途径中可能具有更为重要的作用。

在解析液泡功能的基础上，本研究还聚焦于长春花中异胡豆苷合酶(Strictosidine Synthase, STR1) 及其类似蛋白 (STR2) 的功能鉴定。STR是MIA生物合成途径中的关键酶，负责催化裂环马钱子苷与色胺生成异胡豆苷。通过液泡蛋白质组与代谢组联合分析，筛选获得一具有STR保守结构域的候选蛋白STR2。利用原核表达体系和农杆菌-烟草瞬时表达体系对STR2的酶活性进行了验证，结果表明STR2具有异胡豆苷合酶活性，能够催化生成异胡豆苷。此外，通过亚细胞定位实验，将STR2定位于液泡中，进一步支持其在液泡中参与MIA生物合成的结论。

为解析长春碱合成途径的调控机制，本研究还利用已发表的长春花单细胞转录组数据，筛选了可能参与长春碱合成调控的AP2/ERF转录因子。通过基因共表达分析、系统发育分析及细胞特异性表达模式分析，确定了7个候选转录因子基因。这些候选基因与长春碱合成途径基因在表达模式上存在显著相关性，主要在表皮细胞相关类群中特异性高表达，且在细胞类型表达定位上与部分途径基因相似。这些发现为深入研究AP2/ERF转录因子在长春碱合成途径中的调控作用提供了重要线索。

综上本研究通过多组学方法解析长春花液泡在MIA生物合成途径中的作用，鉴定了具有异胡豆苷合酶活性的STR2蛋白，并筛选到与长春碱合成相关的转录因子。这些发现不仅丰富了对长春花MIA生物合成与调控的理解，而且为通过代谢工程和合成生物学手段提高长春碱等生物碱的产量提供了理论依据，为长春花药用资源的开发与利用提供新的思路和方法。

Abstract

Catharanthus roseus is a perennial medicinal plant with traditional applications in China. It is particularly valued for its ability to synthesize various monoterpenoid indole alkaloids (MIAs) with significant medicinal properties, notably the anticancer drugs vinblastine and vincristine. To elucidate the biosynthetic pathways and regulatory mechanisms of MIAs in C. roseus, this study employed a multi-omics approach to investigate the role of vacuoles in MIA metabolism. We identified a protein, STR2, with strictosidine synthase activity and screened seven key transcription factors associated with vinblastine biosynthesis.

Vacuoles, as essential organelles in plant cells, are involved in osmoregulation, storage, degradation, and the synthesis and accumulation of secondary metabolites. In C. roseus, strictosidine, the common precursor of all MIAs is synthesized in the vacuole, suggesting a critical role for vacuoles in MIA biosynthesis. To explore this further, we first optimized the isolation and purification methods for vacuoles from C. roseus leaf cells using osmotic-thermal shock treatment combined with density gradient centrifugation, successfully obtaining high-quality vacuolar samples. Subsequently, liquid chromatography-tandem mass spectrometry (LC-MS/MS) and ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) were employed to conduct proteomic and metabolomic analyses of vacuolar and protoplast samples.

Proteomic analysis revealed that vacuoles were enriched with proteins related to metabolism, transport, and cellular detoxification, which play crucial roles in maintaining vacuolar function and MIA biosynthesis. Additionally, differentially expressed proteins associated with ion transport, hydrolase activity, and transporter function were identified, suggesting their involvement in the synthesis, transport, and accumulation of MIA precursors. Metabolomic analysis further elucidated the composition of vacuolar metabolites. Comparative analysis of metabolites in vacuoles and protoplasts demonstrated that vacuoles accumulated high levels of alkaloids, phenolic acids, lipids, and flavonoids. Notably, indole alkaloids such as vindoline and catharanthine were significantly more enriched in vacuoles than in protoplasts. These findings not only confirm the role of vacuoles as storage sites for MIAs but also suggest their broader functional significance in MIA biosynthesis.

Building on the functional characterization of vacuoles, this study also focused on identifying the roles of strictosidine synthase (STR1) and its homolog (STR2) in C. roseus. STR is a key enzyme in MIA biosynthesis, catalyzing the condensation of secologanin and tryptamine to form strictosidine. Through integrated vacuolar proteomic and metabolomic analyses, we identified a candidate protein, STR2, containing conserved STR domains. The enzymatic activity of STR2 was validated using prokaryotic expression and Agrobacterium-mediated transient expression in tobacco, confirming its ability to catalyze strictosidine synthesis. Subcellular localization experiments further demonstrated that STR2 is localized to the vacuole, supporting its role in vacuolar MIA biosynthesis.

To decipher the regulatory mechanisms of vinblastine biosynthesis, we screened potential AP2/ERF transcription factors using published single-cell transcriptomic data from C. roseus. Through gene co-expression analysis, phylogenetic analysis, and cell-specific expression profiling, seven candidate transcription factor genes were identified. These genes exhibited significant correlations in expression patterns with vinblastine biosynthetic pathway genes, showing high specificity in epidermal cell populations and overlapping cellular localization with some pathway genes. These findings provide critical insights into the regulatory roles of AP2/ERF transcription factors in vinblastine biosynthesis.

In summary, this study utilized a multi-omics approach to elucidate the role of vacuoles in MIA biosynthesis in C. roseus, identified STR2 as a functional strictosidine synthase, and screened key transcription factors associated with vinblastine biosynthesis. These discoveries enhance our understanding of MIA biosynthesis and regulation, offering a theoretical foundation for improving the production of vinblastine and related alkaloids through metabolic engineering and synthetic biology. Furthermore, this research provides novel strategies for the development and utilization of C. roseus as a medicinal resource.