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Article

Characterization and Expression Analysis of the bHLH Gene Family During Developmental Stages and Under Various Abiotic Stresses in Sanghuangporus baumii

by
Ruipeng Liu
1,
Tingting Sun
1,2,
Pengyu Du
1,
Zengcai Liu
1,
Yawei Li
1,
Xinyu Tong
1 and
Li Zou
1,*
1
College of Forestry, Northeast Forestry University, Harbin 150040, China
2
College of Food Engineering, Harbin University, Harbin 150086, China
*
Author to whom correspondence should be addressed.
Genes 2025, 16(2), 184; https://doi.org/10.3390/genes16020184
Submission received: 11 January 2025 / Revised: 27 January 2025 / Accepted: 28 January 2025 / Published: 2 February 2025
(This article belongs to the Section Microbial Genetics and Genomics)
Browse Figures
Versions Notes

Abstract

:
Background: Basic helix–loop–helix (bHLH) transcription factors (TFs) widely exist in eukaryotic organisms and play a key role in plant growth and development in response to environmental stresses. Sanghuangporus baumii, an important medicinal mushroom known for its anticancer properties, has limited research on the bHLH gene family. Methods: This research utilized the genomic data from S. baumii to identify bHLH family members, and their gene structure, conserved motifs, and phylogenetic relationship were characterized. Additionally, we conducted an analysis of promoter cis-elements and predicted protein interaction networks. We also examined the expression profiles of bHLH genes during different developmental stages and in response to four abiotic stresses: heat, cold, oxidative stress, and heavy metal exposure. Finally, we overexpressed the candidate gene SbbHLH3 in yeast to assess its tolerance to these different stress conditions. Results: A total of 12 SbbHLH genes were identified in S. baumii, and the members of the bHLH gene family displayed a variety of physicochemical characteristics, reflecting their diverse array of functions. Based on homology, the SbbHLH proteins are more closely related to those found in Lentinula edodes and Pleurotus ostreatus. The analysis of promoter cis-elements showed that SbbHLHs contain several elements associated with abiotic stress response, and a network prediction identified 28 bHLH-interacting proteins. Expression pattern analysis revealed that most SbbHLH genes exhibited a positive response to different developmental stages and abiotic stresses. Notably, the overexpression of SbbHLH3 significantly enhanced stress tolerance in yeast. Conclusions: This study provides a comprehensive assessment of the bHLH family in S. baumii, delivering new genetic resources for breeding resistant varieties.

1. Introduction

S. baumii, a precious medicinal mushroom (previously classified as Phellinus baumii and Inonotus baumii), has been utilized as a traditional herbal remedy in China, Korea, Japan, and some other Asian countries for centuries [1]. This fungus typically grows on the trunk of Syringa reticulata and takes approximately 2 to 3 years to develop into yellow mature fruiting bodies [2]. In recent decades, S. baumii has garnered significant attention for its various bioactive compounds, including polysaccharides, triterpenoids, flavonoids, and phenolic compounds, which have important anticancer, antiviral, antioxidant, and anti-inflammatory activities [3,4,5,6]. Due to these properties, the wild resources of S. baumii are being depleted, so many researchers have explored artificial cultivation to increase the yield of this precious fungus [7]. However, S. baumii is susceptible to various abiotic stresses during its lifespan, such as oxidation and extreme temperatures, which lead to the weakening of its growth and development, as well as a significant decline in quality and yield.
Transcription factors (TFs), also known as trans-acting factors, play an important role in improving plant resistance to various environmental stresses [8,9]. TFs can specifically interact with cis-promoter elements of target genes to regulate specific physiological or biochemical processes in cells at the transcript level [10]. Structurally, a typical TF contains four functional domains: a DNA-binding domain, a transcription activation or repression domain, an oligomerization domain, and a nuclear localization domain. TF families, including members of the bHLH, MYB, bZIP, and other TF gene families, represent a significant portion of eukaryotic genomes, and their downstream target genes are extensively involved in numerous essential cellular signaling processes, such as differentiation, metabolism, development, and stress responses [11].
Basic helix–loop–helix (bHLH) proteins constitute one of the largest families of transcription factors and are widely distributed in plants, fungi, and animals [12]. The bHLH domain, which comprises approximately 50 to 60 conserved amino acids, features two primary components: the basic region and the helix–loop–helix (HLH) region. The basic amino acid region, located at the N-terminal and consisting of about 13 amino acids, is primarily responsible for DNA binding. Many bHLH domains recognize and bind to a specific DNA sequence known as the E-box (CANNTG). The HLH region, located at the C-terminal, consists of two α-helices separated by a loop. These α-helices form homo- or heterodimers with other α helices to stabilize the DNA interaction and facilitate transcription [13,14].
To date, most studies of bHLH genes have focused on plant species. The first bHLH gene identified in plants was in maize [15]. Since then, the bHLH gene family has been extensively researched in many plant species, including Arabidopsis thaliana, rice (Oryza sativa), sorghum (Sorghum bicolor), papaya (Carica papaya), Chinese white pear (Pyrus bretschneideri), carrot (Daucus carota), Ginkgo biloba, and Rhododendron delavayi [16,17,18,19,20,21,22]. In contrast, investigations into transcription factors in fungi, particularly mushrooms [23,24], are considerably fewer, with even less focus on bHLH factors. Despite the notable medicinal and economic importance of S. baumii, detailed reports on bHLH transcription factors in this mushroom are lacking, and the specific roles of bHLH genes in this species remain poorly understood. Therefore, it is of great significance to systematically study the bHLH family in S. baumii.
In this research, a total of 12 bHLH (SbbHLH) genes were identified from the reference genome of S. baumii, and phylogenetic analyses were conducted to explore the relationships among these genes. Additionally, we examined the physicochemical properties of the proteins, gene structure, motif composition, three-dimensional structure, and cis-elements in the promoter regions. We also analyzed the expression patterns of all 12 bHLH family members in S. baumii across various developmental stages and in response to abiotic stresses using quantitative real-time PCR (qRT-PCR). Notably, our findings demonstrated that the overexpression of SbbHLH3 enhanced yeast resistance to abiotic stresses. This study may enhance the understanding of the functions of SbbHLH genes under abiotic stress conditions and provide a theoretical foundation for further functional research on the bHLH family in S. baumii.

2. Materials and Methods

2.1. Sample Preparation

S. baumii strain DL101 was collected from S. reticulata and the pure mycelial culture is preserved at the China Center for Type Culture Collection (CCTCC No. M2011137). The mycelium of S. baumii is maintained on potato dextrose agar (PDA) slants, stored at 4 °C, and subcultured every three months. Initially, a seed culture was prepared by incubating it in a rotary shaker incubator at 180 r/min and 25 °C for 8 to 10 days in liquid potato dextrose (PD) medium. Subsequently, a secondary experiment was conducted in 250 mL flasks containing 150 mL of PD medium, inoculated with 8% (v/v) of the seed culture. These flasks were shaken at 180 r/min at 25 °C for 5 days and then exposed to different stress conditions: 50 μM CdCl2 (Aladdin Biochemical Technology Co., Ltd., Shanghai, China), 10 mM H2O2 (Aladdin Biochemical Technology Co., Ltd., Shanghai, China), high temperature (40 °C), and low temperature (4 °C) for 24 to 48 h. Moreover, to examine the expression levels of SbbHLHs at different developmental stages of S. baumii, mycelium was collected at 3, 5, 7, and 9 days, as well as the primordia and young fruiting bodies. Each treatment was performed in triplicate. All samples were subsequently frozen and stored at −80 °C for later analysis.

2.2. Identification of bHLH Genes in the S. baumii Genome

The genome sequence data for S. baumii (NCBI accession number: ASM148141v2) were obtained from the NCBI database (https://www.ncbi.nlm.nih.gov/, accessed on 19 June 2023), and the hidden Markov model (HMM) file of bHLH domain (PF00010) was downloaded from the Pfam (http://pfam.xfam.org/, accessed on 20 June 2023) database. To identify SbbHLH gene family members, HMMER 3.0 software was adopted to search against the S. baumii protein sequences using default settings (E-value cut-off < 1 ×10−5); next, candidate bHLH sequences were further validated using SMART (http://smart.embl-heidelberg.de/, accessed on 21 June 2023) [25,26] and NCBI conserved domain searches. Moreover, we analyzed the physicochemical properties of the SbbHLH proteins, including coding sequence length, isoelectric point (pI), and molecular weight (MW), using the ExPASy Server (http://web.expasy.org/protparam/, accessed on 22 June 2023). The subcellular localization (SL) of the proteins was predicted with the online tool WoLF PSORT (https://www.genscript.com/wolf-psort.html?src=leftbar, accessed on 22 June 2023).

2.3. Characterization of the Structure and Motif of bHLHs

The exon–intron structures of SbbHLH genes were analyzed using the structure display server (GSDS 2.0, https://gsds.gao-lab.org/, accessed on 22 June 2023) [27]. Multiple sequence alignments of SbbHLH proteins were performed using ClustalW with default settings, followed by the construction of a phylogenetic tree based on 1000 bootstrap replicates via the neighbor-joining method using MEGA X 10.0.5 software [28]. The motifs of each deduced SbbHLH protein were identified by MEME (https://meme-suite.org/tools/meme, accessed on 23 June 2023) [29]. TBtools v2.136 software was used to visualize the NJ tree, gene structures, and the motifs of SbbHLH [30].

2.4. Phylogenetic Analysis and Classification of bHLH Gene Family

Schizophyllum commune is regarded as a model species among basidiomycetes, possessing 11 bHLH proteins. According to the classification of bHLHs in S. commune (ScbHLH), all identified SbbHLH genes were categorized into different groups. Multiple sequence alignments of ScbHLH and SbbHLH proteins were conducted using ClustalW, followed by the construction of a phylogenetic tree using the neighbor-joining (NJ) method with 1000 bootstrap replicates in MEGA X software. Moreover, the full-length amino acid sequences of bHLH proteins from S.commune, Agaricus bisporus, Ophiocordyceps sinensis, P. ostreatus, Coprinopsis cinerea, and L. edodes, obtained using a uniform method, were combined with the identified SbbHLH for phylogenetic analysis.

2.5. Homology Modeling of 3D SbbHLH Protein Structures

The secondary structure of the SbbHLH proteins was estimated utilizing SOPMA (https://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_sopma.html, accessed on 23 June 2023). The tertiary structure of the SbbHLHs was constructed and visualized with the help of the SWISS-MODEL platform (https://www.expasy.org/resources/swiss-model, accessed on 24 June 2023).

2.6. Cis-Element Analyses in Promoter Regions of SbbHLH

To infer the potential function of SbbHLH genes, the upstream 2000 bp sequence from the transcription start site of each gene was extracted from the S. baumii genome, and the PlantCare website (https://bioinformatics.psb.ugent.be/webtools/plantcare/html/, accessed on 12 September 2024) was utilized for the prediction of cis-acting elements [31].

2.7. Protein–Protein Interaction Network Prediction

Protein–protein interaction networks were predicted using the STRING database (https://cn.string-db.org/, accessed on 19 September 2024) [32]. A total of 12 SbbHLH protein sequences served as queries, with orthologs from Phellinus noxius (as S. baumii was formerly classified under the genus Phellinus) selected as references. The interaction network for SbbHLH proteins was constructed utilizing the proteins with the highest scores from BLAST analysis.

2.8. Total RNA Extraction, cDNA Reverse Transcription, and qRT-PCR Analysis

All twelve SbbHLH genes were analyzed by qRT-PCR at different developmental stages and under different stress conditions. Total RNA was extracted using the RNAprep Pure plant kit (TIANGEN BIOTECH Co., Ltd., Beijing, China). For reverse transcription, 1 μg of RNA was used as the template to synthesize the first-strand cDNA with the PrimeScript™ RT reagent Kit containing gDNA Eraser (Takara Biomedical Technology Co., Ltd., Dalian, China). The primers for qRT-PCR were designed with Primer Premier 5.0 software (Table S1). The procedure for qRT-PCR was described in previous studies, with the α-tubulin gene used as an internal control [33]. Each experiment was performed in triplicate, and relative gene expression levels were calculated using the 2−(ΔΔCt) method [34].

2.9. Cloning of the SbbHLH3 Gene

Based on the SbbHLH3 nucleic acid sequence obtained, primers were designed with Primer Premier 5.0 software (Table S1) using S. baumii cDNA as a template and high-fidelity enzyme for SbbHLH3 cloning. The purified PCR products were subsequently ligated into the pMD18-T cloning vector (Takara Biomedical Technology Co., Ltd., Dalian, China) for amplification. The sequencing results were verified to match the genomic sequencing data, and these confirmed sequences were then utilized to construct the expression vector.

2.10. Plasmid Construction and Overexpression Analysis of SbbHLH3 in Yeast

Firstly, the pYES2 vector was digested by the single enzyme (HindⅢ, Takara Biomedical Technology Co., Ltd., Dalian, China), and the SbbHLH3 coding sequence was then ligated into the pYES2 vector using MS FastCloning MultiS kit plus (Zhaokui Biomedical Technology Co., Ltd., Beijing, China). The recombinant vector was then transferred into Escherichia coli and sent to Harbin Biotech for sequencing (Genesoul Biotech, Harbin, China). The correctly sequenced recombinant vector was designated as pYES2-SbbHLH3. Both the recombinant vector and the empty pYES2 vector were transformed into INVScI yeast (Weidi BIOTECH Co., Ltd., Shanghai, China) following the manufacturer’s protocol. The transformed yeast cells were then cultured on selective SD-Ura medium (Weidi BIOTECH Co., Ltd., Shanghai, China) at 30 °C for a period of 24 to 48 h. To confirm the accuracy of the sequence, PCR verification was conducted after selecting single colonies, using the primers listed in Table S1 (pYES2-T7/pYES2-CYC1).
To verify the tolerance of SbbHLH3 gene to abiotic stresses, the transformed pYES2-SbbHLH3 yeast and the control yeast (harboring only the pYES2 plasmids) were first incubated in SD-Ura, and then transferred to SC-Ura induction medium containing 2% galactose and incubated at 30 °C for 24 h. Subsequently, the optical density (OD600) was adjusted to 1.0, and the supernatant was discarded after centrifugation. To detect the tolerance of oxidation and heavy metal resistance, the yeast cells were resuspended in 1 mL of SD-Ura medium containing H2O2 solution with a final concentration of 100 mmol L−1, 200 mmol L−1, and 400 mmol L−1, respectively, as well as cadmium chloride solution with a final concentration of 200 μmol L−1, 400 μmol L−1, and 800 μmol L−1. The cultures were then incubated at 30 °C for 24 h. In addition, for freezing and heat stress evaluation, the yeast cells were resuspended in SD-Ura liquid medium and incubated at −20 °C for 24 h and at 50 °C in a water bath for 2 h, respectively. After treatment, the cells were serially diluted (100, 10−1, 10−2, 10−3, and 10−4), and 2 µL drops of the diluted samples were spotted onto the SD-Ura agar medium. The results were evaluated after 2 days of incubation at 30 °C. All experiments were conducted in triplicate.

3. Results

3.1. Identification and Classification of bHLH Genes in S. baumii

A total of 12 bHLH transcription factors were identified in the S. baumii genome (Table S2). To facilitate their identification, they were designated SbbHLH1 through SbbHLH12. The predicted protein lengths of the SbbHLH ranged from 244 (SbbHLH6) to 1254 (SbbHLH8) amino acids. The proteins’ theoretical isoelectric point (pI) values ranged from 5.50 (SbbHLH11) to 9.55 (SbbHLH2), while their estimated molecular weights (MW) were between 26.26 (SbbHLH6) and 137.86 kDa (SbbHLH8). According to subcellular location prediction, most proteins worked in the nucleus, except for SbbHLH5 and SbbHLH8, which were predicted to be located in the extracellular space and mitochondrial compartment, respectively (Table 1). The bHLH proteins from S. baumii were classified as acidic due to their average pI being 6.76. We discovered that all family members are hydrophilic proteins based on Grand Average of Hydropathicity (GRAVY) assessments. Members of the bHLH gene family exhibit a range of physicochemical properties, reflecting their diverse array of functions.

3.2. Gene Structure and Conserved Motif Analysis of SbbHLH

To gain a deeper understanding of the structural components of the SbbHLH genes, we conducted an analysis of their exon–intron structures. The number of introns varied among the SbbHLH genes, ranging from 0 to 5 (Figure 1a,c). The majority of SbbHLH genes (41.67%) contained two introns, while the other genes predominantly had three introns. Notably, SbbHLH8 contained the highest number of introns, totaling five, whereas SbbHLH6 was found to have no introns.
Additionally, we examined the sequence characteristics of the bHLH family members in S. baumii by analyzing the conserved motifs of the 12 SbbHLH proteins using the MEME program. We identified a total of seven specific conserved motifs (Motif 1–7) (Table S3). As shown in Figure 1b, nearly all SbbHLH proteins exhibited the conserved Motif 1 and Motif 2, which were located closely together within the bHLH proteins. Interestingly, we observed that certain motifs were exclusive to specific SbbHLH proteins. For instance, Motifs 3–7 were uniquely present in SbbHLH8 and SbbHLH11, suggesting that these two genes may have distinct functions compared to the other SbbHLH members.

3.3. Multiple Sequence Alignment, Phylogenetic Analysis, and Classification of SbbHLH Genes

For a better understanding of the evolutionary relationship between S. baumii and model basidiomycete Schizophyllum commune, multiple sequence alignment was conducted using ClustalW, focusing on the amino acid sequences of 12 SbbHLH and 11 ScbHLH proteins (Tables S2 and S4). Subsequently, a phylogenetic tree was constructed utilizing the neighbor-joining (NJ) method with a bootstrap value of 1000, implemented in Mega X (Figure 2a). The phylogenetic analysis revealed that the 23 bHLH genes were categorized into nine distinct clades, along with one unclassified group (UC) that encompassed two SbbHLH proteins (SbbHLH8 and SbbHLH12). Notably, none of the ScbHLH proteins were placed in subfamily 8, which exclusively contained two SbbHLH proteins (SbbHLH5 and SbbHLH11), suggesting a potential novel evolutionary pathway for S. baumii. Among the identified subfamilies, subfamily 9 was the most populous, comprising five bHLH members, while the remaining subfamilies each contained two members.
The sequence similarity of the bHLH gene family members from the two species was assessed and visualized using a heatmap (Figure 2b). Several SbbHLHs were tightly clustered with the ScbHLHs (bootstrap support ≥ 70). These SbbHLHs are likely orthologous to the ScbHLHs and may have analogous functions.

3.4. Evolutionary Analysis of bHLH in S. baumii and Several Different Macrofungus Species

To investigate the evolutionary relationships of bHLH proteins in S. baumii and six other macrofungus species (S. commune, A. bisporus, O. sinensis, P. ostreatus, C. cinerea, and L. edodes), we constructed an unrooted neighbor-joining tree incorporating 10 conserved motifs identified by the MEME web server (Figure 3, Table S4). The distribution of SbbHLHs in the phylogenetic tree is notably widespread. With the exception of LebHLH4, all other proteins from the six macrofungi analyzed contained Motifs 1 and 2. Moreover, we observed that the bHLH proteins from L. edodes, P. ostreatus, and S. baumii clustered on the same phylogenetic branch, indicating a generally similar motif composition among them. This suggests a closer evolutionary relationship between SbbHLH proteins and those found in L. edodes and P. ostreatus. Overall, individuals within the same subfamily displayed comparable patterns of conserved motifs, and supporting the reliability of the phylogenetic analysis.

3.5. Cis-Regulatory Element Analysis of SbbHLH Gene Promoters

To better understand the regulatory mechanisms of the SbbHLH gene family in response to abiotic stress, the cis-elements in the promoter region were analyzed using the online database PlantCARE, and the redundant and irrelevant sequences were manually removed before TBtools was used to visualize the findings. As illustrated in Figure 4, the SbbHLH promoter harbors various stress-responsive elements, including TC-rich repeats (involved in defense and stress responsiveness), ARE (anaerobic response element), LTR (low-temperature responsiveness element), and MBS (MYB binding site associated with drought inducibility). Furthermore, the promoter regions also contained several hormone-responsive and growth- and development-related elements, such as the MeJA/CGTCA-motif, abscisic acid/ABRE, salicylic acid/TCA-element, gibberellin/GARE-motif, and auxin/AuxRR-core (Figure 4). Therefore, members of the bHLH gene family in S. baumii are likely to be involved in multiple responses (e.g., stress response) by regulating the upstream elements of transcription factors.

3.6. Analysis of the 3D Structure of SbbHLH Proteins

The three-dimensional (3D) architecture of proteins plays a crucial role in understanding their functions. Consequently, we performed a 3D structural analysis of all 12 SbbHLH proteins using SWISS-MODEL. The findings revealed a significant similarity among several protein pairs, specifically SbbHLH3/4, SbbHLH9/10, and SbbHLH8/11 (Figure S1). By integrating the 3D structures of the SbbHLH proteins, we can gain a deeper insight into the similarities and differences among the family members. This understanding will serve as a valuable reference for further investigations into the functions of SbbHLH proteins in S. baumii.

3.7. In Silico Protein–Protein Interaction of SbbHLH Proteins

To investigate the relationships between SbbHLHs and other proteins, a protein interaction network was constructed by mapping SbbHLHs to homologs from P. noxius (as S. baumii was previously classified under the genus Phellinus) using the STRING online database (Figure 5). The results revealed that 10 SbbHLHs interacted with each other, while the remaining SbbHLH5 and SbbHLH6 showed no interaction with any bHLH proteins. In the network, there were 28 bHLH-interacting proteins, including low temperature requirement protein, steroid regulatory element binding cleavage-activating protein, WD40 domain-containing protein, membrane-bound transcription factor site-2 protease-like protein, and PAS domain-containing protein. These findings provide a valuable reference for further functional studies of the bHLH gene family in S. baumii.

3.8. Expression of SbbHLH Genes at Different Developmental Stages

To investigate the role of bHLH family genes at different developmental stages of S. baumii, we assessed the relative expression levels of all 12 SbbHLH genes in mycelium cultured for 3 to 9 days, as well as in primordia and fruiting bodies, using qRT-PCR. Our analysis revealed distinct expression patterns for the SbbHLH genes across the different developmental stages, indicating that these genes may fulfill diverse regulatory functions. Notably, four genes (SbbHLH1, SbbHLH2, SbbHLH5, and SbbHLH10) exhibited peak transcript levels in mycelium cultured for 5 days, while two genes (SbbHLH6 and SbbHLH7) reached their highest expression in mycelium cultured for 9 days. This suggests a potential involvement of these genes in mycelial development in S. baumii. In contrast, SbbHLH3, SbbHLH4, SbbHLH8, SbbHLH9, SbbHLH11, and SbbHLH12 displayed elevated expression during the primordia and fruiting body stages, leading us to hypothesize that these six SbbHLH genes may be more closely linked to the development of primordia and fruiting bodies (Figure 6).

3.9. Expression Patterns of SbbHLH Genes in Response to Different Stress Treatments

To evaluate the influence of different abiotic stresses on the expression of SbbHLH genes, we conducted a qRT-PCR analysis of 12 SbbHLH members subjected to four types of abiotic stress: H2O2, CdCl2, heat, and cold treatments. The results revealed diverse expression patterns among the SbbHLH genes. Most of the SbbHLH genes exhibited significant upregulation under heat stress, with the exception of SbbHLH11 (Figure 7a). On the contrary, fewer SbbHLH genes were upregulated in response to cold stress compared to heat stress, mainly including SbbHLH3, SbbHLH8, and SbbHLH12 (Figure 7b). Additionally, the expression levels of certain SbbHLH genes varied significantly over time depending on the specific stress treatment. For instance, under CdCl2 stress, the expression of SbbHLH3 and SbbHLH9 was initially elevated but then declined (Figure 7d). Interestingly, some genes displayed contrasting expression patterns under different stress conditions; for example, SbbHLH12, was upregulated in response to heat and CdCl2 treatments, yet downregulated under H2O2 exposure (Figure 7a,c,d). Notably, SbbHLH3 showed a particularly robust response across the various abiotic stresses. Under heat, cold, H2O2, and CdCl2 treatments, SbbHLH3 reached the highest transcript levels (41.7-fold, 20.3-fold, 88.9-fold, and 67.5-fold, respectively) at 12 or 24 h post-induction when compared to the control. Therefore, SbbHLH3 appears to play a crucial role in mediating abiotic stress responses in S. baumii and needs further study.

3.10. Overexpression of SbbHLH3 Enhances Yeast Tolerance to Abiotic Stress

To explore the roles of bHLH family genes in the abiotic stress response of S. baumii, SbbHLH3 was selected for preliminary functional validation based on qRT-PCR expression levels. The coding sequence of SbbHLH3 was cloned into pYES2 plasmids and subsequently transformed into Saccharomyces cerevisiae strains INVSC1. The successful recombinant yeast strains (pYES2-Sb-bHLH3) were confirmed through PCR analysis (Figure S2). Both the positive and control INVSC1 yeast cells were cultured on SD-Ura with different abiotic stress treatments. As shown in Figure 8, under non-stress conditions, there was no notable difference in growth between the control (pYES2) and the transformed yeast (pYES2-Sb-bHLH3), indicating that the overexpression of SbbHLH3 does not affect yeast growth. In contrast, after exposure to heat (50 °C) or cold (−20 °C) stress, the transformed yeast (pYES2-Sb-bHLH3) exhibited superior growth compared to the empty vector control (pYES2).
Furthermore, the pYES2-Sb-bHLH3 yeast strain and the control yeast were cultured under varying concentrations of CdCl2 and H2O2 stress. The results showed that both CdCl2 and H2O2 stress significantly affected yeast cell growth. However, it was evident that the growth of the pYES2-Sb-bHLH3 yeast strain was notably superior to that of the control (Figure 9 and Figure 10). For example, under 200 mM H2O2 stress, the pYES2 control yeast cells did not exhibit any growth after dilution, whereas the pYES2-Sb-bHLH3 yeast continued to grow even after a 1000-fold dilution. This suggests that the overexpression of SbbHLH3 enhances the yeast’s resistance to oxidative stress. In summary, these results indicate that SbbHLH3 may play a beneficial role in the response of fungi to abiotic stress.

4. Discussion

The bHLH gene family has been identified as the second-largest transcription factor family in plants, followed by MYB, and plays a crucial role in various signaling pathways related to growth, development, and stress responses [35]. To date, numerous bHLH genes have been identified in different plant species. Specifically, 164 bHLH TFs were identified in Arabidopsis, 174 in S. bicolor, 180 in rice, 190 in tobacco, 191 in grapes, 146 in carrot, and 208 in maize [16,17,21,36,37]. In this study, we identified 12 bHLH genes in S. baumii. The size of the bHLH gene family in S. baumii (12) is smaller compared to those in plants but is comparable to other fungi, such as 12 in L. edodes, 11 in S. commune, 10 in P. ostreatus, 9 in A. bisporus, 10 in C. cinerea, and 8 in O. sinensis, as determined from their genomic data (Table S4). These findings suggest that the large bHLH gene family in plants has experienced considerable expansion, likely accompanied by subfunctionalization, neofunctionalization, and changes in gene expression patterns and protein–protein interactions.
The protein motif and gene structure analysis indicated that various members of the same subfamily likely share common evolutionary origins and may have similar physiological roles. The number of exons/introns in SbbHLH family members varied from 0 to 5, resembling findings from previous studies. This indicates that during the evolution of S. baumii, the bHLH gene exons and introns underwent processes of loss or gain (Figure 1c). Through phylogenetic analysis with S. commune, we categorized the SbbHLH family proteins into nine distinct subfamilies (Figure 2a). Subcellular localization studies revealed that the majority of SbbHLHs were localized in the cell nucleus (Table 1), aligning with the findings related to bHLH family genes in other organisms [17,38].
The bHLHs can form homodimers or heterodimers and bind to specific cis-acting elements on target genes [39]. In this study, we examined the cis-regulatory elements of SbbHLH gene promoters and identified conserved regulatory modules that are characteristic of various members involved in stress responsiveness, as well as growth- and development-related elements in the promoter regions. Notable motifs identified include TC-rich repeats, ARE, LTR, and MBS motifs. The findings suggest that SbbHLHs may play a role in growth and stress resistance in S. baumii. Additionally, predicted protein interaction networks provide a theoretical framework for exploring the physiological roles of different bHLH family proteins. The physical interactions among proteins within the network imply that SbbHLHs could participate in a range of cellular processes. However, SbbHLH5 and SbbHLH6 did not demonstrate interactions with other SbbHLH proteins, suggesting that these two genes may be involved in distinct metabolic pathways.
Gene expression patterns can offer valuable insights into gene function. The bHLH gene family displays considerable variation in abundance among different organisms and tissues, contributing to diverse physiological roles [36]. In our research, the SbbHLH genes displayed distinct expression patterns. Some SbbHLHs showed peak transcript levels at specific stages of reproductive development, suggesting that they may regulate particular developmental processes by influencing various target genes. Our findings revealed that the expression levels of SbbHLH3, SbbHLH8, and SbbHLH9 were dramatically upregulated (over 20-fold in qPCR results) in the primordia or fruiting bodies, and thus we speculated that these three genes participate in development processes of S. baumii (Figure 6). Additionally, numerous studies have indicated that bHLH genes are crucial in responding to abiotic stress [21,38,39,40]. The increased expression of SbbHLHs under heat stress suggests their extensive involvement in the heat stress response. Among the identified 12 SbbHLHs, SbbHLH3 and SbbHLH9 exhibited enhanced expression during the reproductive stages and under various abiotic stress conditions, highlighting their multifunctional roles in S. baumii (Figure 7). Notably, SbbHLH3 exhibited particularly high expression levels across different treatments. Consequently, heterologous expression in yeast was utilized in subsequent experiments to explore the preliminary functions of SbbHLH3. The findings demonstrated that the overexpression of SbbHLH3 significantly improved the tolerance of yeast cells to various stresses (Figure 8, Figure 9 and Figure 10). This suggests that SbbHLH3 may play a positive role in enhancing the resilience of S. baumii to abiotic stresses.
This study has some limitations, such as the inability to confirm protein–protein interactions in vivo. In addition, the specific mechanism by which the SbbHLH3 gene contributes to the resistance of S. baumii was not fully investigated, even though the bHLH gene family was identified. Future research should aim to explore the resistance-related genes in S. baumii, and analyze the relationship between its medicinal properties and resistance. Furthermore, gaining insights into the molecular characteristics of S. baumii will enhance its economic potential.

5. Conclusions

In summary, a thorough analysis identified 12 SbbHLH genes in the S. baumii genome. These genes underwent various evaluations, including physicochemical properties, phylogenetic relationships, conserved motifs, gene structure, cis-regulatory element, protein–protein interactions, and expression patterns. Notably, the expression analysis and heterologous expression assays in yeast indicated that SbbHLH3 may be involved in development and abiotic stress responses in S. baumii. This study provides detailed insights into the functional mechanisms and expression profiles of SbbHLHs in S. baumii, laying the theoretical groundwork for future research on the molecular mechanisms of SbbHLHs.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/genes16020184/s1, Figure S1: The three-dimensional (3D) architecture of SbbHLH proteins; Figure S2: PCR gel electrophoresis identification results for the recombinant yeast INVSC1 (pYES2-Sb-bHLH3) strains. Table S1: Primers used for this study; Table S2: List of the 12 SbbHLH genes identified in this study; Table S3: Analysis and distribution of conserved motifs in S. baumii bHLH proteins; Table S4: List of the bHLH genes of six macrofungus species identified in this study; Table S5: Analysis and distribution of conserved motifs in bHLH proteins of seven macrofungus species.

Author Contributions

Writing—original draft preparation, R.L. and T.S.; Data curation, P.D., Z.L. and T.S.; Validation, X.T. and Y.L.; Writing—review and editing, L.Z. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the National Natural Science Foundation of China (32171792), the National Natural Science Foundation of Heilongjiang Province (LH2022C054).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in this study are included in the article/Supplementary Materials. Further inquiries can be directed to the corresponding author.

Conflicts of Interest

The authors declare no conflicts of interest.

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Genes 16 00184 g001
Figure 1. Characterization of S. baumii bHLH genes. (a) Phylogenetic analysis of SbbHLH proteins from S. baumii. (b) Conserved motifs of SbbHLH proteins. (c) Gene structures of SbbHLH genes. Introns are shown as gray lines.
Figure 1. Characterization of S. baumii bHLH genes. (a) Phylogenetic analysis of SbbHLH proteins from S. baumii. (b) Conserved motifs of SbbHLH proteins. (c) Gene structures of SbbHLH genes. Introns are shown as gray lines.
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Figure 2. The phylogenetic tree among bHLH domains of S. baumii and S. commune. (a) The phylogenetic tree was derived using the NJ method in MEGA X. (b) Heatmap of sequence similarity among members of the bHLHs of S. baumii and S. commune.
Figure 2. The phylogenetic tree among bHLH domains of S. baumii and S. commune. (a) The phylogenetic tree was derived using the NJ method in MEGA X. (b) Heatmap of sequence similarity among members of the bHLHs of S. baumii and S. commune.
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Figure 3. Phylogenetic relationship and motif composition of the bHLH proteins from S. baumii with six different macrofungus species (S. commune, A. bisporus, O. sinensis, P. ostreatus, C. cinerea, and L. edodes).
Figure 3. Phylogenetic relationship and motif composition of the bHLH proteins from S. baumii with six different macrofungus species (S. commune, A. bisporus, O. sinensis, P. ostreatus, C. cinerea, and L. edodes).
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Figure 4. Cis-acting element analysis of SbbHLH promoters.
Figure 4. Cis-acting element analysis of SbbHLH promoters.
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Figure 5. Prediction of SbbHLH-interacting protein networks in S. baumii.
Figure 5. Prediction of SbbHLH-interacting protein networks in S. baumii.
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Figure 6. Gene expression of 12 SbbHLH genes determined by qRT-PCR during different developmental stages of S. baumii. (The data shown are mycelium cultured for 3~9 days: 3d, 5d, 7d, and 9d; primordium; and fruit body.) The error bars represent the standard error of the mean of three technical replicates. Lowercase letters indicate significant differences among treatment means (α = 0.05, LSD). The dashed line indicates that the relative expression level of the gene reaches “1”, making it easy to compare with the control.
Figure 6. Gene expression of 12 SbbHLH genes determined by qRT-PCR during different developmental stages of S. baumii. (The data shown are mycelium cultured for 3~9 days: 3d, 5d, 7d, and 9d; primordium; and fruit body.) The error bars represent the standard error of the mean of three technical replicates. Lowercase letters indicate significant differences among treatment means (α = 0.05, LSD). The dashed line indicates that the relative expression level of the gene reaches “1”, making it easy to compare with the control.
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Genes 16 00184 g007aGenes 16 00184 g007b
Figure 7. The expression profile of 12 SbbHLH genes in S. baumii under abiotic stresses as evaluated by qRT-PCR: (a) heat treatment; (b) cold treatment; (c) H2O2 treatment; and (d) CdCl2 treatment. The error bars represent the standard error of the mean of three technical replicates. Lowercase letters indicate significant differences among treatment means (α = 0.05, LSD). The dashed line indicates that the relative expression level of the gene reaches “1”, making it easy to compare with the control.
Figure 7. The expression profile of 12 SbbHLH genes in S. baumii under abiotic stresses as evaluated by qRT-PCR: (a) heat treatment; (b) cold treatment; (c) H2O2 treatment; and (d) CdCl2 treatment. The error bars represent the standard error of the mean of three technical replicates. Lowercase letters indicate significant differences among treatment means (α = 0.05, LSD). The dashed line indicates that the relative expression level of the gene reaches “1”, making it easy to compare with the control.
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Figure 8. Effects of SbbHLH3 expression on the survival of S. cerevisiae strains with heat or cold or no treatment.
Figure 8. Effects of SbbHLH3 expression on the survival of S. cerevisiae strains with heat or cold or no treatment.
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Figure 9. Effects of SbbHLH3 expression on the survival of S. cerevisiae strains supplemented with different concentrations of CdCl2.
Figure 9. Effects of SbbHLH3 expression on the survival of S. cerevisiae strains supplemented with different concentrations of CdCl2.
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Figure 10. Effects of SbbHLH3 expression on the survival of S. cerevisiae strains supplemented with different concentrations of H2O2.
Figure 10. Effects of SbbHLH3 expression on the survival of S. cerevisiae strains supplemented with different concentrations of H2O2.
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Table 1. The 12 bHLH transcription factors identified in S. baumii.
Table 1. The 12 bHLH transcription factors identified in S. baumii.
No.GeneGene IDCDS Length
(bp)		Protein Size
(aa)		Molecular Weight (kDa)pIPredicted
Localization
1SbbHLH1A7U60_g182091230332.596.48nuclear
2SbbHLH2A7U60_g1898188162667.939.55nuclear
3SbbHLH3A7U60_g2241258986289.356.38nuclear
4SbbHLH4A7U60_g2862114037941.105.97nuclear
5SbbHLH5A7U60_g3672171657161.278.03extracellular
6SbbHLH6A7U60_g465473524426.266.44nuclear
7SbbHLH7A7U60_g6711137445748.076.08nuclear
8SbbHLH8A7U60_g720537651254137.866.39mitochondrial
9SbbHLH9A7U60_g724791530433.747.91nuclear
10SbbHLH10A7U60_g7248105034938.756.23nuclear
11SbbHLH11A7U60_g801934981165125.745.50nuclear
12SbbHLH12A7U60_g8101279693196.976.17nuclear
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Liu, R.; Sun, T.; Du, P.; Liu, Z.; Li, Y.; Tong, X.; Zou, L. Characterization and Expression Analysis of the bHLH Gene Family During Developmental Stages and Under Various Abiotic Stresses in Sanghuangporus baumii. Genes 2025, 16, 184. https://doi.org/10.3390/genes16020184

AMA Style

Liu R, Sun T, Du P, Liu Z, Li Y, Tong X, Zou L. Characterization and Expression Analysis of the bHLH Gene Family During Developmental Stages and Under Various Abiotic Stresses in Sanghuangporus baumii. Genes. 2025; 16(2):184. https://doi.org/10.3390/genes16020184

Chicago/Turabian Style

Liu, Ruipeng, Tingting Sun, Pengyu Du, Zengcai Liu, Yawei Li, Xinyu Tong, and Li Zou. 2025. "Characterization and Expression Analysis of the bHLH Gene Family During Developmental Stages and Under Various Abiotic Stresses in Sanghuangporus baumii" Genes 16, no. 2: 184. https://doi.org/10.3390/genes16020184

APA Style

Liu, R., Sun, T., Du, P., Liu, Z., Li, Y., Tong, X., & Zou, L. (2025). Characterization and Expression Analysis of the bHLH Gene Family During Developmental Stages and Under Various Abiotic Stresses in Sanghuangporus baumii. Genes, 16(2), 184. https://doi.org/10.3390/genes16020184

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