Archive > Volume 18 Issue 1 > 2026,18(1):200-211. DOI:https://doi.org/10.1016/j.chmed.2025.05.005 Prev Next

灵芝bHLH转录因子的全基因组鉴定及功能分析

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Comprehensive genomic identification and functional analysis of bHLH transcription factors in Ganoderma lucidum

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摘要:

目的：碱性螺旋-环-螺旋（bHLH）转录因子在真菌生长、发育及次生代谢调控中具有重要作用，但其在灵芝（Ganoderma lucidum）三萜类成分（灵芝酸）生物合成中的功能尚不明确。本研究旨在解析灵芝bHLH家族基因在灵芝发育过程中对灵芝酸合成的调控机制。方法：首先通过隐马尔可夫模型搜索和双向比对完成灵芝全基因组范围内GlbHLHs的鉴定；结合理化性质、基因结构和系统进化分析，整合灵芝不同发育阶段的转录组与代谢组数据筛选候选基因；进一步利用酵母单杂交和双荧光素酶报告系统验证其调控功能。结果：在灵芝中鉴定到11个GlbHLHs成员，其启动子区富集激素与非生物胁迫响应元件。尽管单体灵芝酸在特定发育阶段和组织中积累模式差异显著，但整体上菌盖中总灵芝酸含量始终高于菌柄。所有GlbHLHs在灵芝从原基期至成熟期的全菌中高表达，其中GlbHLH5和GlbHLH7在各阶段表达量最高，并与灵芝酸合成通路关键基因高度相关。功能验证表明，GlbHLH5可激活羊毛甾醇合酶启动子的P2区，而GlbHLH7可激活鲨烯环氧酶和鲨烯合酶的启动子活性。结论：与植物相比，灵芝bHLH家族成员数量较少但均呈现高表达特性，其中GlbHLH5和GlbHLH7对灵芝酸合成具有激活作用。本研究为灵芝酸合成的分子调控机制提供了新见解，为提升灵芝酸产量及优化其药用价值奠定理论基础。

Abstract:

Objective: The basic helix-loop-helix (bHLH) transcription factors (TFs) are pivotal in regulating fungal growth, development, and secondary metabolism. However, the knowledge about the Ganoderma lucidum bHLHs (GlbHLHs) in ganoderic acid (GA) biosynthesis of G. lucidum was limited. This study aimed to explore the functions of bHLH genes in ganoderic acid biosynthesis during G. lucidum growth development. Methods: First, the genome-wide identification of GlbHLHs was performed through Hidden Markov model searches and Two-way blast. Furthermore, through physicochemical properties, gene structure, and phylogenetic analysis, as well as combining the transcriptome and metabolome data from different developmental stages of G. lucidum, candidate GlbHLHs were screened. Subsequently, their regulatory roles in ganoderic acid biosynthesis were explored using yeast one-hybrid and dual luciferase reporter assays. Results: A total of 11 GlbHLH members were characterized in G. lucidum. The upstream promoter regions of these genes enriched hormones and abiotic stress responsive elements. Although individual ganoderic acid monomers demonstrated marked differences in accumulation patterns across specific growth phases and tissue types, overall, the total GA content was consistently higher in caps than in stipes throughout development. In addition, all GlbHLHs exhibited high expression in whole G. lucidum from the primordium to maturation stages. Among them, GlbHLH5 and GlbHLH7 showed the highest expression in any stage and highly correlated with key genes associated with GA pathway. Functional validation through dual-luciferase assays and yeast one-hybrid experiments had demonstrated that GlbHLH5 activated the P2 region of the lanosterol synthase promoter, while GlbHLH7 activated the promoters of squalene epoxidase and squalene synthase. Conclusion: Compared to plants, G. lucidum harbored a small number of bHLH members but all high expression in any stages. Additionally, GlbHLH5 and GlbHLH7 with the highest expression among GlbHLHs showed activation in regulating the biosynthesis of GA. These results provide a theoretical reference for further research on ganoderic acid regulation in G. lucidum, and thereby providing a molecular foundation for enhancing ganoderic acid yield to optimize the medicinal value of G. lucidum.

关键词:

bHLH转录因子;灵芝酸;灵芝;基因组鉴定;代谢组学

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We are grateful for the instrument support provided by the large-scale instrument sharing platform of the Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences. This research received funding from the Scientific and Technological Innovation Project of China Academy of Chinese Medical Sciences (No. CI2021A04008), National Key Research and Development Project (No. 2023YFC3504104), Hangzhou Joint Fund of the Zhejiang Provincial Natural Science Foundation of China (No. LHZSZ24H280003), Technology Major Program on Agricultural New Variety Breeding (No. 2021C02073), Central Guiding Local Science and Technology Development Fund Project (No. 2024ZY01009).

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