脱落酸和乙烯利对白桦丛生苗三萜积累及其合成途径基因的调控作用

Effects of abscisic acid and ethephon on triterpenoid accumulation and biosynthetic pathway genes in Betula platyphylla

摘要

【目的】白桦树皮中五环三萜类化合物具有重要的药理活性，但其天然来源有限，制约其进一步开发利用。白桦三萜类化合物的积累受细胞工程技术与外源激素诱导的共同调控。本研究旨在探讨外源添加脱落酸（ABA）和乙烯利对白桦组培苗中三萜类化合物积累及其生物合成途径关键基因表达的调控效应。【方法】以白桦丛生苗为材料，于培养第 7 天分别进行不同浓度 ABA 和乙烯利处理，分别利用高效液相色谱、比色法及荧光定量 PCR 技术，测定三萜类化合物含量、抗氧化酶活性以及相关基因表达变化。【结果】（1）10 µmol/L ABA 处理后显著促进白桦丛生苗中三萜及组分的积累，其中总三萜、白桦脂醇和齐墩果酸含量分别达到 82.43、24.61 和 33.13 mg/g，分别比对照提高了 46.29%、28.73% 和 22.78%。5 mmol/L 乙烯利处理后总三萜、白桦脂醇和齐墩果酸含量最高，分别为 78.53、26.54 和 35.84 mg/g，分别比对照提高了 39.07%、54.04% 和 27.02%。（2）两种激素诱导后三萜合成基因（ HMGR、 BPY 及 DS）及胁迫响应相关基因（ CYP450 与 CP）均有不同程度的上调，其中 HMGR 最为显著，抗氧化酶（CAT、SOD、APX 和 PAL）活性得到增强。（3）茉莉酸（JA）和水杨酸（SA）信号途径中的关键基因显著受到 ABA 和乙烯利的诱导，其中 JAZ1/2 和 NPR1 较对照表现为上调表达， JAZ3/4、 NPR3 表现为下调表达，JA 信号途径重要转录因子 MYC2、 bHLH44/42 和 TGA2/5/7 均表现为上调表达。【结论】10 μmol/L ABA 或 5 mmol/L 乙烯利可有效促进白桦丛生苗中三萜物质的积累，同时可以激活白桦防御酶系统，诱导 JA、SA 信号途径相关基因及三萜合成相关基因上调表达。本研究为利用植物细胞工程技术提高白桦三萜类化合物产量提供了理论依据和技术参考。

Abstract

[Objective] Pentacyclic triterpenoids in birch bark exhibit significant pharmacological activity; however, their natural availability is limited, thereby restricting their further development and utilization. The accumulation of birch triterpenoids is jointly regulated by plant cell engineering technologies and exogenous hormone induction. This study aimed to investigate the regulatory effects of exogenous abscisic acid (ABA) and ethephon on triterpenoid accumulation and the expression of key genes involved in triterpenoid biosynthesis in birch tissue-cultured plantlets. [Method] Clustered birch plantlets were used as experimental materials. Different concentrations of ABA and ethephon were applied on the 7th day of culture. High-performance liquid chromatography (HPLC), colorimetric assays, and quantitative real-time PCR (RT-qPCR) were employed to determine triterpenoid contents, antioxidant enzyme activities, and the expression levels of related genes. [Result] (1) Treatment with 10 μmol/L ABA significantly promoted the accumulation of triterpenoids and their components in birch plantlets. The contents of total triterpenoids, betulin, and oleanolic acid reached 82.43, 24.61, and 33.13 mg/g, respectively, representing increases of 46.29%, 28.73%, and 22.78% compared with the control. Under ethephon treatment at 5 mmol/L, the highest contents of total triterpenoids, betulin, and oleanolic acid were 78.53, 26.54, and 35.84 mg/g, respectively, corresponding to increases of 39.07%, 54.04%, and 27.02% over the control. (2) Both hormone treatments upregulated the expression of triterpenoid biosynthesis-related genes (HMGR, BPY, and DS) and stress-responsive genes (CYP450 and CP) to varying degrees, with HMGR showing the most pronounced response. In addition, the activities of antioxidant enzymes, including CAT, SOD, APX, and PAL, were significantly enhanced. (3) Key genes in the jasmonic acid (JA) and salicylic acid (SA) signaling pathways were markedly induced by ABA and ethephon treatments. Compared with the control, JAZ1/2 and NPR1 were upregulated, whereas JAZ3/4 and NPR3 were downregulated. Furthermore, important transcription factors in the JA signaling pathway, including MYC2, bHLH44/42, and TGA2/5/7, were significantly upregulated. [Conclusion] Treatment with 10 μmol/L ABA or 5 mmol/L ethephon effectively promoted triterpenoid accumulation in birch plantlets. These treatments also activated the antioxidant defense system and induced the expression of genes involved in JA and SA signaling pathways as well as triterpenoid biosynthesis. This study provides a theoretical basis and technical reference for enhancing triterpenoid production in birch through plant cell engineering approaches.

关键词

白桦 / 脱落酸 / 乙烯利 / 三萜类化合物 / 生物合成基因 / JA/SA 信号通路

Key words

Betula platyphylla / abscisic acid / ethephon / triterpenoids / biosynthetic genes / JA/SA signaling pathway

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基金资助

国家自然科学基金项目(32371840)

黑龙江省自然科学基金重点项目(ZD2023C004)

黑龙江春燕创新团队计划(CYCX24023)