丹参RBOH基因家族特征及盐胁迫响应分析

doi:10.13560/j.cnki.biotech.bull.1985.2026-0593

摘要/Abstract

摘要：

目的呼吸爆发氧化酶同源物（respiratory burst oxidase homologs, RBOHs）是植物活性氧（ROS）生成的关键限速酶，广泛参与植物逆境胁迫的调控过程。探究丹参RBOHs基因在盐胁迫应答及活性成分积累中的调控特征，为深入解析SmRBOHs基因功能、阐明其调控丹参活性成分积累的分子机制提供理论支撑。方法以拟南芥RBOH蛋白序列为参考，系统鉴定丹参RBOHs基因家族成员，全面解析其序列特征、进化关系、启动子顺式作用元件与转录因子结合位点，并构建其蛋白互作调控网络；结合转录组数据筛选与丹参活性成分生物合成通路基因共表达的SmRBOHs。通过盐胁迫处理试验，利用RT-qPCR等技术，系统分析SmRBOHs表达模式与ROS含量、丹酚酸和丹参酮积累的相关性。结果从丹参基因组中鉴定到6个SmRBOHs，系统进化分析可将其划分为3个亚组，其基因启动子区富含盐胁迫、脱落酸（abscisic acid, ABA）等逆境响应顺式元件和转录因子结合位点。该家族基因具有组织表达特异性，可差异化响应ABA信号，且与丹酚酸、丹参酮合成通路基因存在共表达关系。盐胁迫下，SmRBOHs各成员表达模式分化明显，其中SmRBOH2表达量与H₂O₂、丹参酮含量呈负相关，与丹酚酸积累呈正相关，其余家族成员则呈相反变化趋势。结论丹参RBOHs广泛参与植物激素介导的生理调控与盐胁迫应答过程，且其转录表达受多类转录因子调控。丹参或可通过ABA介导的SnRK2-RBOH信号模块，差异化调控丹酚酸与丹参酮的生物合成，进而参与盐胁迫适应性应答。

关键词: 丹参, RBOH基因家族, 盐胁迫, 表达模式, 丹酚酸, 丹参酮, H₂O₂, 相关分析

Abstract:

Objective Respiratory burst oxidase homologs (RBOHs) are key rate-limiting enzymes that generate reactive oxygen species (ROS) in plants and play critical roles in stress responses. This study aims to investigate the regulatory characteristics of SmRBOHs under salt stress and their effects on active ingredient accumulation, thereby providing a theoretical basis for further functional analysis of SmRBOHs and for elucidating the molecular mechanisms underlying active component accumulation in Salvia miltiorrhiza. Method Using Arabidopsis thaliana RBOH protein sequences as a reference, we systematically identified members of the SmRBOH gene family, comprehensively analyzed their sequence characteristics, evolutionary relationships, and promoter cis-elements and transcription factor binding sites, and constructed a protein-protein interaction network. Transcriptome data were then used to screen for SmRBOHs co-expressed with genes involved in the biosynthetic pathways of salvianolic acids and tanshinones. Under salt stress treatments, real-time quantitative PCR (RT-qPCR) and other techniques were applied to analyze the correlations between SmRBOH expression levels, ROS content, and the accumulation of salvianolic acids and tanshinones. Result A total of six SmRBOHs were identified and classified into three subgroups based on phylogenetic analysis. Their promoter regions contained abundant cis-elements associated with abiotic stresses such as salt stress and abscisic acid (ABA), as well as transcription factor binding sites. The SmRBOHs exhibited tissue-specific expression patterns and differential responses to ABA. Additionally, these genes were co-expressed with key genes involved in the biosynthetic pathways of salvianolic acids and tanshinones. Under salt stress, the SmRBOH members displayed distinct expression patterns. Among them, SmRBOH2 expression was negatively correlated with H₂O₂ and tanshinone contents, but positively correlated with salvianolic acid accumulation. The other members showed opposite trends. Conclusion SmRBOHs are widely involved in hormone-mediated physiological regulation and salt stress responses in S. miltiorrhiza, and their transcriptional expression is modulated by various transcription factors. S. miltiorrhiza may regulate the biosynthesis of salvianolic acids and tanshinones through the ABA-mediated SnRK2-RBOH signaling module, thereby participating in adaptive responses to salt stress.

Key words: Salvia miltiorrhiza, RBOH gene family, salt stress, expression pattern, salvianolic acids, tanshinones, H₂O₂, correlation analysis

李贺勤, 闫恒宇, 袁语锌, 段炎炎, 李彩丽, 单成钢, 江绪文. 丹参RBOH基因家族特征及盐胁迫响应分析[J]. 生物技术通报, doi: 10.13560/j.cnki.biotech.bull.1985.2026-0593.

LI He-qin, YAN Heng-yu, YUAN Yu-xin, DUAN Yan-yan, LI Cai-li, SHAN Cheng-gang, JIANG Xu-wen. Characterization of the RBOH Gene Family and Analysis of Its Salt Stress Responses in Salvia miltiorrhiza[J]. Biotechnology Bulletin, doi: 10.13560/j.cnki.biotech.bull.1985.2026-0593.

图/表 7

图1 SmRBOHs的保守结构域（A）、基因基序（B）和基因结构（C）

Fig. 1 Conserved domain (A), gene motif (B), and gene structure (C) of SmRBOHs

图2 丹参、拟南芥、水稻、番茄、黄芩和墨西哥鼠尾草RBOH蛋白的系统进化关系

Fig. 2 Phylogenetic relationships of RBOH proteins from Salvia miltiorrhiza, Arabidopsis thaliana, Oryza sativa, Solanum lycopersicum, Scutellaria baicalensis, and Salvia divinorum

图3 SmRBOHs顺式作用元件及转录因子结合位点和蛋白互作分析A：顺式作用元件类型；B：顺式作用元件数量；C：转录因子结合位点；D：蛋白互作分析

Fig. 3 Analysis of cis-acting elements, transcription factor binding sites, and protein-protein interactions of the SmRBOHsA: Types of cis-acting elements. B: Numbers of cis-acting elements. C: Transcription factor binding sites. D: Protein-protein interaction analysis

图4 SmRBOHs表达模式A：SmRBOHs在不同组织中的表达；B：SmRBOHs与丹参酮生物合成途径基因响应ABA的共表达模式；C：SmRBOHs与丹酚酸生物合成途径基因响应ABA的共表达模式

Fig. 4 Expression patterns of SmRBOHsA: Expression of SmRBOHs in different tissues. B: Co-expression pattern of SmRBOHs and genes involved in the biosynthesis pathway of tanshinone in response to ABA. C: Co-expression pattern of SmRBOHs and genes involved in the biosynthesis pathway of salvianolic acid in response to ABA

图5 盐胁迫下丹参盆栽苗表型、根系H2O2含量和SmRBOHs表达特征A‒B：盐处理的丹参盆栽苗（A）和根系H2O2含量（B）；C‒H：盐处理的丹参根系SmRBOHs的相对表达量。*P<0.05，下同

Fig. 5 Phenotypes of pot seedlings, root H2O2 content, and SmRBOHs expression of S. miltiorrhiza under salt stressA‒B: S. miltiorrhiza pot seedlings(A) and root H2O2 content (B) under salt stress. C‒H: Relative expression of SmRBOHs in roots of salt‑treated S. miltiorrhiza seedlings. *P<0.05. The same below

图6 盐胁迫对丹参根系丹酚酸（A）和丹参酮（B）含量的影响

Fig. 6 Effect of salt stress on the contents of salvianolic acid (A) and tanshinone (B) in the roots of S. miltiorrhiza

图7 不同处理丹参根系SmRBOHs表达水平与H2O2含量、丹酚酸含量和丹参酮含量的相关性分析CA：咖啡酸；RA：迷迭香酸；SaA：丹酚酸A；SaB：丹酚酸B；DT-I：二氢丹参酮I；CT：隐丹参酮；T-I：丹参酮I；T-IIA：丹参酮IIA。**P<0.01

Fig. 7 Correlation analysis of SmRBOHs expression in roots of S. miltiorrhiza under different treatments with H2O2, salvianolic acid, and tanshinone contentsCA: Caffeic acid. RA: Rosmarinic acid. SaA: Salvianolic acid A. SaB: Salvianolic acid B. DT-I: Dihydrotanshinone I. CT: Cryptotanshinone. T-I: Tanshinone I. T-IIA: Tanshinone IIA. **P<0.01

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