檀香烯合酶基因启动子克隆及其活性分析

Cloning and its activity analysis of santalene synthase gene (SaSSy) promoter from Santalum album

摘要

【目的】檀香精油的主要活性成分为檀香醇，其生物合成关键酶--檀香烯合酶（SaSSy）基因的转录调控机制尚不清晰。本研究旨在克隆并初步鉴定檀香 SaSSy基因的启动子活性，探索其转录调控特征。【方法】采用 Tail-PCR 技术克隆檀香 SaSSy基因 ATG 上游 1 437 bp 的启动子序列；通过生物信息学分析其顺式作用元件及潜在转录因子结合位点，并进行 KEGG 通路富集分析；利用烟草叶片瞬时转化实验验证启动子活性；使用 100 µmol/L 茉莉酸甲酯（MeJA）处理檀香愈伤组织，通过 RT-qPCR 和 GUS 活性检测分析 SaSSy表达及启动子响应；通过启动子截断实验鉴定核心转录调控区域。【结果】成功克隆获得 SaSSy基因 1 437 bp 的启动子序列，其顺式作用元件包含光、激素及胁迫响应元件。预测发现与之互作的转录因子主要富集于“植物激素信号转导”“MAPK 信号通路−植物”“植物−病原互作”及“植物昼夜节律”通路。烟草瞬时转化证实该启动子具有转录激活活性。MeJA 处理可显著诱导檀香愈伤组织中 SaSSy基因的表达上调及该启动子驱动的 GUS 活性增强。截断实验表明，−264 bp 的核心启动子区域（P SaSSy-D4）即可驱动转录，其活性与克隆的全长启动子相当。【结论】本研究揭示了 SaSSy基因启动子的基本特征，证实其表达受 MeJA 正向调控，并确定了包含关键元件的−264 bp 核心启动子区域。该启动子的 MeJA 诱导特性为探索通过外源信号分子提升檀香精油生物合成提供了实验依据，也为进一步解析檀香醇合成的转录调控网络提供了候选靶点。

Abstract

[Objective] The primary active component of sandalwood essential oil is santalol, yet the transcriptional regulatory mechanism of key biosynthetic enzyme gene santalene synthase (SaSSy) remains unclear. This study aimed to clone and preliminarily characterize the promoter of SaSSy gene in Santalum album, and to explore its transcriptional regulatory features. [Method] The 1 437 bp promoter sequence upstream of ATG start codon of SaSSy gene was cloned from Santalum album using Tail-PCR. Cis-regulatory element analysis was performed to identify functional motifs. Transcription factor binding sites were predicted, and KEGG enrichment analysis was conducted to explore associated regulatory pathways. The transcriptional activity of the promoter was verified via transient expression assays in tobacco leaves. Sandalwood calli were treated with 100 µmol/L methyl jasmonate (MeJA), and SaSSy expression was analyzed by RT-qPCR. GUS activity driven by the promoter was measured to assess MeJA responsiveness. Truncated promoter versions were constructed to identify functional regions. [Result] A 1 437 bp promoter sequence of SaSSy was successfully cloned, containing light-, hormone-, and stress-responsive cis-elements. Transcription factor binding site prediction and KEGG analysis revealed enrichment in pathways related to plant hormone signaling, MAPK signaling, plant-pathogen interaction and circadian rhythm. The promoter demonstrated transcriptional activity in tobacco transient assays. MeJA treatment significantly upregulated SaSSy expression and enhanced promoter-driven GUS activity. A truncated 264 bp core promoter region (P SaSSy-D4) retained full transcriptional activity, comparable to full-length promoter. [Conclusion] The present study reveals fundamental characteristics of P SaSSy, confirming that its expression is positively regulated by MeJA, and identifies a core promoter region (−264 bp) containing key cis-acting elements. The MeJA-inducible characteristics of P SaSSy provide experimental evidence for enhancing sandalwood essential oil biosynthesis through exogenous signalling molecules, and offer a candidate target for further elucidating the transcriptional regulatory network of santalol synthesis.

关键词

檀香烯合酶 / SaSSy启动子 / 遗传转化烟草 / 茉莉酸甲酯诱导

Key words

santalene synthase / SaSSy promoter / transgenic Nicotiana tabacum / MeJA induction

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基金资助

国家自然科学基金项目(32060358)

广西自然科学基金项目(2019GXNSFAA185005)