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

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 G. lucidum bHLHs ( GlbHLHs ) in ganoderic acid (GA) biosynthesis of Ganoderma 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 GlbHLH s 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.