The Ganoderma lucidum strain (G. lucidum CGMCC5.26) used in this experiment was purchased from the Institute of Microbiology, Chinese Academy of Sciences, the DH5a competent cells were purchased from Vazyme, and AH109 competent cells were purchased from Shanghai Weidi Biotechnology Co., Ltd. The pGBKT7 vector was presented by Dr. Bingcong Xing from Zhejiang A&F University.

Fungal complete medium (CYM) and fungal protoplast cell wall regeneration medium (MYG) were used to cultivate G. lucidum. CYM medium was composed of 1% maltose, 2% glucose, 0.2% yeast extract, 0.2% peptone, 0.05% MgSO₄⋅7H₂O, 0.05% K₂HPO₄ (Zhou et al., 2014). MYG medium was composed of 1% glucose, 0.5% maltose, 0.5% yeast powder, 2% agar powder, dissolved in mannitol. LB medium: 1% tryptone, 0.5% yeast powder, 1% NaCl, 2% agar powder (solid), pH = 7.0.

The primers were designed according to the CDS region of the GlbHLH5 gene (Supplementary Table 1) and synthesized by Sangon Biotech. The total RNA of Ganoderma lucidum hyphae was extracted according to the instructions of the column-type fungal total RNA extraction and purification kit (Sangon Biotech, Shanghai, China). Agarose gel electrophoresis was used to detect the integrity of the total RNA, and a micro-ultraviolet spectrophotometer was used to detect the concentration and purity. Following the instructions of the PrimeScript™ RT Master Mix kit (Sangon Biotech, Shanghai, China), the total RNA was reversed into cDNA which was further used as a template for PCR amplification of the coding region of the GlbHLH5 gene, and the PCR amplification system was as follows: PrimeSTAR Max Premix (2X) 25 μL, GlbHLH5-F 2.5 μL, GlbHLH5-R 2.5 μL, cDNA template 1.0 μL, ddH₂O 19 μL. PCR was performed according to the manufacture’s instruction of PrimeSTAR^® Max DNA Polymerase (Takara, Japan) using the following protocol: 98^°C, 5 min, 1 cycle; 98^°C, 10 s, 55^°C, 30 s, 72^°C, 1 min, 30 cycles; 72^°C, 5 min. After the PCR products were detected by 1.5% agarose gel electrophoresis, they were sent to Zhejiang Youkang Biotechnology Co., Ltd. for sequencing.

A bHLH gene GlbHLH5 (GenBank No. MZ436906.1) was obtained by comparing the G. lucidum genome database¹ and the Pfam database (number: PF00010) (Chen S. et al., 2012; Qian et al., 2013; Finn et al., 2016; Chen X. et al., 2021). Online analysis software ORF Finder and Conserved Domain-search of the NCBI database were used to analyze the open reading frame (ORF) and conserved domains of the gene. ProtParam was performed to analyze the physicochemical properties of the GlbHLH5 encoded protein. At last, highly similar protein sequences of other species were downloaded from the NCBI protein database, and a phylogenetic tree was constructed to perform the phylogenetic analysis by MEGA 7.0. PlantCARE was used to perform online promoter cis-acting element analysis on the nucleic acid sequences of 2 000 bp upstream of three key genes HMGR (GenBank No. EU263989) (Shang et al., 2008), SQS (GenBank No. DQ494674) (Zhao et al., 2007), and LS (GenBank No. GQ169528) (Shang et al., 2010) in the triterpenoid synthesis pathway of Ganoderma lucidum, and TBtools software was used for visual display.

The sequenced gene was translated, and the properties of the protein encoded by the GlbHLH5 gene were analyzed. Using the method of homologous recombination, the GlbHLH5 gene was integrated into the pET-32a vector, and the recombinant plasmid was transferred into Escherichia coli DH5a competent cells, cultured at 37°C overnight, and a single colony was picked for PCR verification and sent to the company for sequencing. Plasmid extraction was performed on the correctly sequenced recombinant-positive strains, and the plasmid DNA was transferred into E. coli BL21 (DE3) competent cells, cultured overnight at 37°C, and single colonies were picked and inoculated in Amp-resistant LB liquid medium, and then cultured at 37°C and 200 r⋅min^–1 until the bacterial density (OD600) reached 0.8. Subsequently, 0.5 mmol⋅L^–1. IPTG was added to induce 12 h at 16, 25, and 37°C, and the bacterial liquid was collected and detected by SDS-PAGE.

To investigate the transcriptional activation of GlbHLH5, pGBKT7 (pBD) was used to construct yeast one-hybrid system pBD-GlbHLH5 vector by EcoRI and BamHI. After the sequencing results were correct, the plasmid pBD-GlbHLH5 and the positive control pGBKT7 were transformed into yeast AH109, respectively. The constructed plasmid was coated on the defect medium SD/-Trp, cultured at 30°C for 3 days, and then transferred to SD/-Trp/-His/-Ade + x-a-Gal for color reaction.

The 50 μmol L^–1 MeJA was added to the fermentation broth of G. lucidum cultured for 5 days, and no MeJA treatment was used as a control. The triterpenoid content of G. lucidum was determined at 0, 2, 4, 6, 12, 24, and 48 h after MeJA induction. The relative expression of GlbHLH5 was determined by qRT-PCR, and the response of the key enzyme genes in the triterpenoid’s biosynthetic pathways to MeJA induction was also investigated. Each experiment was repeated in three replicates, and the data are all expressed as mean ± SD (Gharari et al., 2020).

The GlbHLH5 sequence and overexpression vector pBARGPEI were analyzed, and primers containing BamHI and EcoRI sites were designed (Supplementary Table 1). The pBARGPEI was digested with BamHI and EcoRI, and recovered and purified for homologous recombination with GlbHLH5 containing homologous arms (Song et al., 2021). The recombinant product was transferred into E. coli DH5a competent cells and cultured overnight at 37°C. Single colonies were picked for PCR verification of bacterial solution and sent to the company for sequencing. The plasmid was extracted from the bacterial solution with correct sequencing. The strains WT, pBARGPEI-GFP and pBARGPEI-GlbHLH5-GFP were inoculated in the fungal medium containing CaCl₂, and the sterilized coverslip was inserted into the medium at 1 cm around the inoculation block. After the mycelia grew on the coverslip, the coverslip was stained with DAPI, and rinsed with PBS for several times. The positions of GFP protein, GlbHLH5-GFP fusion protein and nucleus in cells were observed by inverted fluorescence microscope.

According to the GlbHLH5 sequence and the overexpression vector pBARGPEI, primers containing restriction sites BamHI and EcoRI (Supplementary Table 1) were designed. PBARGPEI was digested with BamHI and EcoRI with double restriction enzymes, recovered and purified, and then homologously recombined with GlbHLH5 containing homology arms. The recombined product was transferred to E. coli DH5a competent cells, cultured overnight at 37°C, and the single colonies were picked on Amp resistant plates. Then, bacterial liquid PCR and sequencing were performed to verify whether the recombinant vector was successfully constructed. At last the recombinant plasmids were transformed into protoplast of G. lucidum.

According to pSilent-1 plasmid and GlbHLH5 silencing sequences, suitable restriction sites were selected to design the primers (Supplementary Table 1). The pSilent-1 and GlbHLH5 silencing sequences were double digested with Xhol and HindIII, and the digested products were ligated and transformed and sequenced to form the intermediate vector pS-1. Then, the intermediate vector and GlbHLH5 reverse silencing sequence were doubly digested with SphI and KpnI, and the GlbHLH5 reverse silencing sequence was insert into the downstream of the IT sequence of the vector to finally construct the pSilent-GlbHLH5 recombinant plasmid with an inverted repeat “hairpin structure.”

The overexpression and silencing vectors constructed above were transferred into G. lucidum protoplasts by a PEG-mediated method (Liu and Friesen, 2012), and the transformants were screened by hygromycin (G-Clone, Beijing, China) resistance, and the well-growing strains were further identified by colony PCR and the detection of the GlbHLH5 gene expression.

The expression levels of GlbHLH5, HMGR, SQS, and LS genes were determined by qRT-PCR with 18S rRNA as the internal reference gene, and the primers used were shown in Supplementary Table 1. qRT-PCR was performed according to the manufacture’s instruction of ChamQ Universal SYBR qPCR Master Mix (Vazyme, Nanjing, China) using the following protocol: 95°C, 30 s, 1 cycle; 95°C, 10 s, 60°C, 30 s, 40 cycles. Salkowski reaction was firstly used for qualitative detection of triterpenoid content in G. lucidum, and further quantitative determination was conducted by the vanillin-perchloric acid chromogenic method (Chen Z. et al., 2021).

GraphPad Prism 8.0 software was used for statistical analysis, in which the qRT-PCR results used 18S rRNA as an internal reference, and the relative expression level of genes was calculated by the 2^{–△△ Ct} method, and then the t-test was used to analyze the significance of the difference. “*” means the difference is significant (P < 0.05) and “^**” means the difference is extremely significant (P < 0.01).

The cDNA of G. lucidum was used as a template for PCR amplification. After sequencing, the full length of the Ganoderma bHLH transcription factor cDNA was 1008 bp, and the sequence had been uploaded to the NCBI database. The obtained gene sequence number was MZ436906.1 and named GlbHLH5. The relative molecular weight of GlbHLH5 is 85113.08, the theoretical PI value is 4.98, and it could encode a protein with a molecular weight of about 36.58 kDa. The SMART analysis and multiple comparisons of amino acids showed that it contained an HLH domain composed of 51 amino acids. The phylogenetic analysis indicated that GlbHLH5 was closely related to Ganoderma sinensis transcription factor GsPIL31529.1 (Figure 1).

On-line protein analysis showed that there were three secondary structures of GlbHLH5 gene. The proportion of α-helix (blue), extended strand (red) and random coil (purple) were 26.57, 5.97, and 67.46%, respectively (Figure 2A-a). SWISS-MODEL was used to predict the three-dimensional structure of GlbHLH5 protein online. The three-dimensional structure was mainly α-helix and random coil, which was consistent with the predicted secondary structure of GlbHLH5 protein (Figure 2A-b). Subsequently, the complete coding sequence of GlbHLH5 was cloned into the pET-32a vector and transformed into DH5a competent cells. The recombinant plasmid pET-32a-GlbHLH5 was sequenced and extracted. Then, the recombinant plasmid pET-32a-GlbHLH5 was transformed into E. coli BL21 (DE3) competent cells. The empty vector pET-32a was used as the control. The recombinant cells were collected after IPTG induction for 12 h. After centrifugation, the cells were suspended in Tris-HCl for ultrasonic fragmentation and lysis. The supernatant was taken for SDS-PAGE detection after denaturation. The results showed that the pET-32a-GlbHLH5 strain inducted by IPTG for 12 h could all express the protein with a size of about 36.58 KDa (Figure 2B). With the increase of induction temperature, the protein expression levels firstly increased and then decreased, and the expression level of the target protein was the highest under the induction condition of 25°C.

The complete coding sequence of GlbHLH5 gene was cloned into the pGBKT7 (pBD) vector using the yeast single hybridization system, and the constructed pBD-GlbHLH5 vector was identified by colonies PCR and sequencing. The sequenced recombinant plasmid was transformed into the yeast AH109 strain to observe its growth on the Kan-resistant plate. The results showed that pBD empty vector and pBD-GlbHLH5 transformants could grow well on SD/-Trp plates, while only pBD-GlbHLH5 could grow well on SD/-Trp/-His/-Ade solid medium supplemented with 3-AT (aminotriazole) (Figure 3). Because AT is His synthase inhibitor, it can reduce the leakage expression of His synthase in yeast AH109 strain and improve the selection conditions. The above results show that GlbHLH5 has strong transcriptional activation activity.

Compared with the control group, adding exogenous MeJA significantly increased the content of Ganoderma triterpenoids, and the difference was extremely significant after 12 h of induction (P < 0.01) (Figure 4A). To further clarify whether GlbHLH5 participates in the regulation of Ganoderma triterpenoids biosynthesis by responding to jasmonic acid signals, qRT-PCR was used to determine the response of GlbHLH5 and key genes HMGR, SQS, LS in the biosynthetic pathway of Ganoderma triterpenoids to MeJA induction. After 4 h of induction with 50 μmol L^–1 MeJA, the expression of GlbHLH5 increased significantly (P < 0.01), which was twice that of the control group (Figure 4B); compared to the control group, the expression of HMGR increased significantly (P < 0.05) after 4 h of induction, and reached its peak at 12 h of induction (Figure 4C); after 6 h of induction, the expression of SQS was extremely significantly (P < 0.01) higher than the control group, the expression level reached its peak at 12 h after induction (Figure 4D); the expression of LS was extremely significantly (P < 0.01) higher than the control group after 4 h of induction (Figure 4E). To sum up, the relative expression levels of the three key genes in the triterpenoids biosynthetic pathway all reached their peak values at 12 h after MeJA induction, which was consistent with the induced expression pattern of GlbHLH5. At the same time, the correlation analysis showed that the expression of GlbHLH5 has a very significant (P < 0.01) positive correlation with the three key genes HMGR, SQS, and LS (Figure 4F). Promoter cis-acting elements analysis of the three key genes shown that they all contained multiple G-box, which is the binding site of bHLH transcription factors. It was suggested that GlbHLH5 transcription factor might be involved in triterpenoid biosynthesis in G. lucidum by regulating HMGR, SQS, and LS gene expressions. The regulatory effect of GlbHLH5 in Ganoderma triterpenoids biosynthesis will be further analyzed and verified by transgenic technology.

The online software WoLF PSORT was used to analyze GlbHLH5 transcription factor, and the prediction showed that it was mainly located in the nucleus. The fluorescent protein was observed by laser confocal microscope, and the pBARGPEI-GFP was used as the control group, and the pBARGPEI-GlbHLH5-GFP was used as the experimental group. The results showed that in the presence of Ca²⁺, the nucleus of pBARGPEI-GlbHLH5-GFP strain had obvious fluorescence, while that of WT strain had no fluorescence (Figure 5). It was indicated that GlbHLH5 transcription factor mainly located in the nucleus of G. lucidum, which was consistent with the predicted results, indicating that GlbHLH5 plays a role in the nucleus.

Transgenic Ganoderma lucidum strains overexpressing GlbHLH5 were generated by a PEG-mediated genetic transformation system. The overexpression lines were selected by hygromycin resistance. PCR amplification was also conducted to verify the positive transformants. A 580 bp gpd promoter of the vector was amplified in the GlbHLH5-overexpressed positive transformants but not in the wild strain. It was demonstrated in Figure 6A that GlbHLH5 overexpressed G. lucidum strains could grow well on the hygromycin plate. As for the biosynthesis of Ganoderma triterpenoids, it was shown that the color developing of three overexpression lines (OE-GlbHLH5-1, OE-GlbHLH5-2, and OE-GlbHLH5-3) was deeper than that of the wild type in Salkowski qualitative reaction, visually manifesting the triterpenoid content of the three overexpression lines might be higher than that of the wild type. Further quantitative analysis by the vanillin-perchloric acid chromogenic method revealed that the triterpenoid content of OE-GlbHLH5-1, OE-GlbHLH5-2, and OE-GlbHLH5-3 lines increased by 45, 37, and 21%, respectively (Figure 6B) compared with the wild type. qRT-PCR confirmed that much higher expression levels of GlbHLH5 were observed in the three overexpression lines. Moreover, the transcript levels of the key genes HMGR, SQS, and LS in the biosynthetic pathways of Ganoderma triterpenoids were all upregulated in GlbHLH5 overexpression lines compared with those in the wild type. As shown in Figure 6B, the relative expressions of HMGR, SQS, and LS reached the highest in line OE-GlbHLH5-1, which were 3.16, 3.23, and 2.62 fold of the wild type, respectively. Last but not the least, the variations of the pathway genes transcription were consistent with the accumulation of Ganoderma triterpenoids, and the expression levels of GlbHLH5 as well, indicating that overexpression of GlbHLH5 transcription factor could increase the triterpenoid content by activating the biosynthetic pathways of triterpenoids in G. lucidum.

Three stably inherited GlbHLH5 silenced strains were obtained through PEG-mediated multiple-resistance screening on Hyg plates, and the Hyg gene of the vector was amplified from the GlbHLH5-silenced positive transformants, yet not from the wild strain. As shown in Figure 6B, the relative expression levels of GlbHLH5 gene in the three selected GlbHLH5 silencing lines (Si-GlbHLH5-1, Si-GlbHLH5-2, and Si-GlbHLH5-3) were significantly lower than those in wild type (P < 0.05). The effect of silencing GlbHLH5 on the Ganoderma triterpenoids synthesis and the key genes expressions of biosynthetic pathway was completely opposite to that of GlbHLH5 overexpression. Compared with the wild type, the accumulation of Ganoderma triterpenoids decreased in GlbHLH5-silenced lines, as the Salkowski qualitative reaction showed that the color developing of the three silencing strains was shallower than that of the wild type (Figure 7A). Further quantitative analysis by the vanillin-perchloric acid chromogenic method showed that the triterpenoids content of Si-GlbHLH5-1, Si-GlbHLH5-2, and Si-GlbHLH5-3 decreased by 24, 31, and 19%, respectively. To further verify the function of GlbHLH5 on regulating Ganoderma triterpenoids synthesis, the key genes expressions of their biosynthetic pathways were determined by quantitative RT-PCR. It was revealed that silencing of GlbHLH5 gene leaded to down-regulation of the biosynthetic pathways of Ganoderma triterpenoids. The relative expression levels of HMGR, SQS, and LS genes in lines Si-GlbHLH5-1, Si-GlbHLH5-2 and Si-GlbHLH5-3 were all significantly lower than those in wild type (P < 0.01) (Figure 7B), and the variation in triterpenoids biosynthetic pathway gene transcription was generally consistent with GlbHLH5. The results showed that silencing GlbHLH5 transcription factor could reduce the biosynthesis of triterpenoids in G. lucidum, which further proved that GlbHLH5 transcription factor was a transcription factor involved in the regulation of Ganoderma triterpenoids biosynthesis.