Objective  Cordyceps guangdongensis, a rare edible-medicinal fungus in China, has been approved as a new resource food, with a broad prospect of industrial application. The fruitingbodies of this fungus are rich in nutrients and have significant active effect. The protoplast preparation and genetic transformation methods of Cordyceps guangdongensis were established in order to provide references for new variety breeding and gene function research of this fungus.

Method  Protoplasts were separated from the mycelia of C. guangdongensis by various enzymes. During the process of protoplasts preparation, the enzyme combinations, enzyme digestion time and temperature, regeneration medium of protoplasts and resistance marker were selected by using single-factor analyses. Plasmid pCAMBIA1300 was used as transformation fragment to establish a genetic transformation system, and the concentration of spermidine, PEG addition interval and resistance marker addition time during PEG-mediated protoplast transformation were also screened by single-factor analyses. The knockout fragment of Zn2Cys6-type transcription factor CgPRo1 in C. guangdongensis was used as transformation fragment to verify the transformation system.

Result  The optimal condition for protoplast preparation was mycelium treated with lytic enzyme and driselase (1:1) for 5.5 h at 28 ℃. The highest protoplast regeneration rate of C. guangdongensis was observed on TB₃ medium, followed by 0.6 mol/L KCl-PDA. During the transformation process, adding 5 mmol/L spermidine, PEG at an interval of 10 min and screening medium with 250 μg/mL hygromycin after protoplast regeneration for 3 days showed the optimal transformation efficiency. In addition, three CgPro1 knockout mutants and three heterozygotes were obtained through the established transformation system. Phenotypic analysis results showed that CgPro1 was involved in the fruit body development of C. guangdongensis.

Conclusion  In the study, the optimized protoplast preparation method and PEG-mediated protoplast transformation system for C. guangdongensis were established. The techniques and procedures described will lay a solid foundation for future researches on gene function and protein localization, and also provide important application value in genetic breeding of in C. guangdongensis.