Optimization of Protoplast Preparation and Establishment of PEG-Mediated Genetic Transformation Method in <i>Cordyceps cicadae</i>
<i>Cordyceps cicadae</i> (<i>C. cicadae</i>) is an important edible medicinal fungus; however, owing to its wild growth and lack of genome annotation, construction of a stable genetic transformation system in <i>C. cicadae</i> is greatly limited, impeding the exte...
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2025-03-01
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| author | Haikun Qi Haihua Ruan Tao Wu Hongyang Zhang Rui Dong Yanjun Jiang |
| author_facet | Haikun Qi Haihua Ruan Tao Wu Hongyang Zhang Rui Dong Yanjun Jiang |
| author_sort | Haikun Qi |
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| description | <i>Cordyceps cicadae</i> (<i>C. cicadae</i>) is an important edible medicinal fungus; however, owing to its wild growth and lack of genome annotation, construction of a stable genetic transformation system in <i>C. cicadae</i> is greatly limited, impeding the extensive exploitation of <i>C. cicadae</i> in industry. Here, we successfully established an efficient plasmid transformation method within protoplasts of <i>C. cicadae</i> by PEG mediation using pCas9-EGFP as a marker plasmid. In order to overcome low transformation efficiency and acquire sufficient protoplasts for transformation, the influence of enzyme species, enzymatic hydrolysis time, enzymatic hydrolysis temperature, and fungal age on protoplast preparation were analyzed sequentially, and the optimal conditions for protoplast preparation were determined as follows: 2-day-old <i>C. cicadae</i> mycelia with 1.5% lywallzyme hydrolysis at 34 °C for 5 h. Our results indicate that no less than 5.1 × 10<sup>7</sup> CFU/mL protoplasts could be acquired. Additionally, five osmotic pressure stabilizers including potassium chloride (KCl), sodium chloride (NaCl), glucose, mannitol, and sucrose were employed to enhance the regeneration of protoplasts, among which sucrose exhibited the highest regeneration rate of 10.43%. The transformation efficiency of plasmid was 37.3 CFU/µg DNA. On this basis, a genetic transformation method was successfully constructed, laying the foundation for further gene editing and metabolic engineering of <i>C. cicadae</i>. |
| format | Article |
| id | doaj-art-b84cd048ce94427a8f0d5c045083e9c3 |
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| issn | 2309-608X |
| language | English |
| publishDate | 2025-03-01 |
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| series | Journal of Fungi |
| spelling | doaj-art-b84cd048ce94427a8f0d5c045083e9c32025-08-20T01:48:46ZengMDPI AGJournal of Fungi2309-608X2025-03-0111321910.3390/jof11030219Optimization of Protoplast Preparation and Establishment of PEG-Mediated Genetic Transformation Method in <i>Cordyceps cicadae</i>Haikun Qi0Haihua Ruan1Tao Wu2Hongyang Zhang3Rui Dong4Yanjun Jiang5Tianjin Key Laboratory of Food Science and Biotechnology, College of Biotechnology and Food Science, Tianjin University of Commerce, Tianjin 300134, ChinaTianjin Key Laboratory of Food Science and Biotechnology, College of Biotechnology and Food Science, Tianjin University of Commerce, Tianjin 300134, ChinaTianjin Key Laboratory of Food Science and Biotechnology, College of Biotechnology and Food Science, Tianjin University of Commerce, Tianjin 300134, ChinaTianjin Key Laboratory of Food Science and Biotechnology, College of Biotechnology and Food Science, Tianjin University of Commerce, Tianjin 300134, ChinaTianjin Key Laboratory of Food Science and Biotechnology, College of Biotechnology and Food Science, Tianjin University of Commerce, Tianjin 300134, ChinaSchool of Chemical Engineering and Technology, Hebei University of Technology, Tianjin 300130, China<i>Cordyceps cicadae</i> (<i>C. cicadae</i>) is an important edible medicinal fungus; however, owing to its wild growth and lack of genome annotation, construction of a stable genetic transformation system in <i>C. cicadae</i> is greatly limited, impeding the extensive exploitation of <i>C. cicadae</i> in industry. Here, we successfully established an efficient plasmid transformation method within protoplasts of <i>C. cicadae</i> by PEG mediation using pCas9-EGFP as a marker plasmid. In order to overcome low transformation efficiency and acquire sufficient protoplasts for transformation, the influence of enzyme species, enzymatic hydrolysis time, enzymatic hydrolysis temperature, and fungal age on protoplast preparation were analyzed sequentially, and the optimal conditions for protoplast preparation were determined as follows: 2-day-old <i>C. cicadae</i> mycelia with 1.5% lywallzyme hydrolysis at 34 °C for 5 h. Our results indicate that no less than 5.1 × 10<sup>7</sup> CFU/mL protoplasts could be acquired. Additionally, five osmotic pressure stabilizers including potassium chloride (KCl), sodium chloride (NaCl), glucose, mannitol, and sucrose were employed to enhance the regeneration of protoplasts, among which sucrose exhibited the highest regeneration rate of 10.43%. The transformation efficiency of plasmid was 37.3 CFU/µg DNA. On this basis, a genetic transformation method was successfully constructed, laying the foundation for further gene editing and metabolic engineering of <i>C. cicadae</i>.https://www.mdpi.com/2309-608X/11/3/219<i>Cordyceps cicadae</i>protoplastsoptimizationgenetic transformation |
| spellingShingle | Haikun Qi Haihua Ruan Tao Wu Hongyang Zhang Rui Dong Yanjun Jiang Optimization of Protoplast Preparation and Establishment of PEG-Mediated Genetic Transformation Method in <i>Cordyceps cicadae</i> Journal of Fungi <i>Cordyceps cicadae</i> protoplasts optimization genetic transformation |
| title | Optimization of Protoplast Preparation and Establishment of PEG-Mediated Genetic Transformation Method in <i>Cordyceps cicadae</i> |
| title_full | Optimization of Protoplast Preparation and Establishment of PEG-Mediated Genetic Transformation Method in <i>Cordyceps cicadae</i> |
| title_fullStr | Optimization of Protoplast Preparation and Establishment of PEG-Mediated Genetic Transformation Method in <i>Cordyceps cicadae</i> |
| title_full_unstemmed | Optimization of Protoplast Preparation and Establishment of PEG-Mediated Genetic Transformation Method in <i>Cordyceps cicadae</i> |
| title_short | Optimization of Protoplast Preparation and Establishment of PEG-Mediated Genetic Transformation Method in <i>Cordyceps cicadae</i> |
| title_sort | optimization of protoplast preparation and establishment of peg mediated genetic transformation method in i cordyceps cicadae i |
| topic | <i>Cordyceps cicadae</i> protoplasts optimization genetic transformation |
| url | https://www.mdpi.com/2309-608X/11/3/219 |
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