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) Background: Sanghuangporus baumii, a valuable medicinal fungus, has limited studies on its gene function due to the lack of a genetic transformation system. (2) Methods: This study aimed to establish an efficient Agrobacterium tumefaciens-mediated transformation (ATMT) system for S. baumii. This study involved cloning the promoter (glyceraldehyde-3-phosphate dehydrogenase, gpd) of S. baumii, reconstructing the transformation vector, optimizing the treatment of receptor tissues, and inventing a new method for screening positive transformants. (3) Results: The established ATMT system involved replacing the CaMV35S promoter of pCAMBIA-1301 with the gpd promoter of S. baumii to construct the pCAMBIA-SH-gpd transformation vector. The vectors were then transferred to A. tumefaciens (EHA105) for infection. This study found that the transformation efficiency was higher in the infection using pCAMBIA-SH-gpd vectors than using pCAMBIA-1301 vectors. The mycelia of S. baumii were homogenized for 20 s and collected as the genetic transformation receptor. After 20 min of co-culture and 48 h of incubation in 15 mL PDL medium at 25 °C, new colonies grew. (4) Conclusions: These colonies were transferred to PDA medium (hygromycin 4 μg/mL, cefotaxime 300 μg/mL), and the transformation efficiency was determined to be 33.7% using PCR.