Enhancement of efficient molecular biology tools for Tremella fuciformis spore

Tremella fuciformis, one of the most approved mushrooms worldwide, possesses both edible and medicinal value. However, low transformation efficiency and high false positive rate, long verification period, and low expression of heterologous proteins hinder further genetic research. In this study, a polyethylene glycol-mediated genetic transformation system was established by using the homologous resistance marker carboxin in T. fuciformis. By optimizing the protoplast quality and transformation conditions, the transformation efficiency reached 8.23 CFU/μg, and the positive rate reached 64.95%. Moreover, Driselase-Heating-Freezing screening system was developed for rapid transformants verification, which took only 35–45 min and made the colony PCR 100% accurate. Considering the rationality of tRNA use frequency in heterologous genes, the coordinated relative synonymous codon usage (RSCU) strategy was adopted to recode the heterologous gene sequence, which ensured the protein expression level. This study comprehensively enhances molecular biology tools in T. fuciformis, and lays a foundation for further development.