Optimization of Protoplast Preparation Conditions in <i>Lyophyllum decastes</i> and Transcriptomic Analysis Throughout the Process
Protoplasts are essential tools for genetic manipulation and functional genomics research in fungi. This study systematically optimized protoplast preparation conditions and examined transcriptional changes throughout the preparation and regeneration processes to elucidate the molecular mechanisms u...
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MDPI AG
2024-12-01
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| Series: | Journal of Fungi |
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| Online Access: | https://www.mdpi.com/2309-608X/10/12/886 |
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| author | Xiaobin Li Ying Qin Yufei Kong Samantha Chandranath Karunarathna Yunjiang Liang Jize Xu |
| author_facet | Xiaobin Li Ying Qin Yufei Kong Samantha Chandranath Karunarathna Yunjiang Liang Jize Xu |
| author_sort | Xiaobin Li |
| collection | DOAJ |
| description | Protoplasts are essential tools for genetic manipulation and functional genomics research in fungi. This study systematically optimized protoplast preparation conditions and examined transcriptional changes throughout the preparation and regeneration processes to elucidate the molecular mechanisms underlying the formation and regeneration of protoplasts in <i>Lyophyllum decastes</i>. The results indicated an optimal protoplast yield of 5.475 × 10<sup>6</sup> cells/mL under conditions of fungal age at 10 days, digestion time of 2.25 h, enzyme concentration of 2%, and digestion temperature of 28 °C. The Z5 medium supplemented with <i>L. decastes</i> mycelial extract achieved a high regeneration rate of 2.86. RNA-seq analysis revealed 2432 differentially expressed genes (DEGs) during protoplast formation and 5825 DEGs during regeneration. Casein kinase I, cytochrome P450 (CYP52), and redox-regulated input receptor (PEX5) were significantly upregulated during the protoplast stage, while β-1,3-glucan synthase (SKN1), chitin synthase (CHS2), hydrophobin-1, and hydrophobin-2 showed significant upregulation during the protoplast regeneration phase. These findings provide a reference for the efficient preparation and regeneration of protoplasts and offer new insights into the molecular mechanisms of protoplast formation and cell wall regeneration in fungi. |
| format | Article |
| id | doaj-art-6aa42662f3cc4ce49645706caa07cf33 |
| institution | DOAJ |
| issn | 2309-608X |
| language | English |
| publishDate | 2024-12-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Journal of Fungi |
| spelling | doaj-art-6aa42662f3cc4ce49645706caa07cf332025-08-20T02:53:17ZengMDPI AGJournal of Fungi2309-608X2024-12-01101288610.3390/jof10120886Optimization of Protoplast Preparation Conditions in <i>Lyophyllum decastes</i> and Transcriptomic Analysis Throughout the ProcessXiaobin Li0Ying Qin1Yufei Kong2Samantha Chandranath Karunarathna3Yunjiang Liang4Jize Xu5College of Agriculture, Yanbian University, Yanji 133002, ChinaCollege of Agriculture, Jilin Agricultural Science and Technology University, Jilin 132000, ChinaCollege of Agriculture, Yanbian University, Yanji 133002, ChinaCenter for Yunnan Plateau Biological Resources Protection and Utilization, Qujing Normal University, Qujing 655011, ChinaCollege of Agriculture, Yanbian University, Yanji 133002, ChinaCollege of Agriculture, Yanbian University, Yanji 133002, ChinaProtoplasts are essential tools for genetic manipulation and functional genomics research in fungi. This study systematically optimized protoplast preparation conditions and examined transcriptional changes throughout the preparation and regeneration processes to elucidate the molecular mechanisms underlying the formation and regeneration of protoplasts in <i>Lyophyllum decastes</i>. The results indicated an optimal protoplast yield of 5.475 × 10<sup>6</sup> cells/mL under conditions of fungal age at 10 days, digestion time of 2.25 h, enzyme concentration of 2%, and digestion temperature of 28 °C. The Z5 medium supplemented with <i>L. decastes</i> mycelial extract achieved a high regeneration rate of 2.86. RNA-seq analysis revealed 2432 differentially expressed genes (DEGs) during protoplast formation and 5825 DEGs during regeneration. Casein kinase I, cytochrome P450 (CYP52), and redox-regulated input receptor (PEX5) were significantly upregulated during the protoplast stage, while β-1,3-glucan synthase (SKN1), chitin synthase (CHS2), hydrophobin-1, and hydrophobin-2 showed significant upregulation during the protoplast regeneration phase. These findings provide a reference for the efficient preparation and regeneration of protoplasts and offer new insights into the molecular mechanisms of protoplast formation and cell wall regeneration in fungi.https://www.mdpi.com/2309-608X/10/12/886functional genomicsfungiprotoplast regenerationRNA-seq |
| spellingShingle | Xiaobin Li Ying Qin Yufei Kong Samantha Chandranath Karunarathna Yunjiang Liang Jize Xu Optimization of Protoplast Preparation Conditions in <i>Lyophyllum decastes</i> and Transcriptomic Analysis Throughout the Process Journal of Fungi functional genomics fungi protoplast regeneration RNA-seq |
| title | Optimization of Protoplast Preparation Conditions in <i>Lyophyllum decastes</i> and Transcriptomic Analysis Throughout the Process |
| title_full | Optimization of Protoplast Preparation Conditions in <i>Lyophyllum decastes</i> and Transcriptomic Analysis Throughout the Process |
| title_fullStr | Optimization of Protoplast Preparation Conditions in <i>Lyophyllum decastes</i> and Transcriptomic Analysis Throughout the Process |
| title_full_unstemmed | Optimization of Protoplast Preparation Conditions in <i>Lyophyllum decastes</i> and Transcriptomic Analysis Throughout the Process |
| title_short | Optimization of Protoplast Preparation Conditions in <i>Lyophyllum decastes</i> and Transcriptomic Analysis Throughout the Process |
| title_sort | optimization of protoplast preparation conditions in i lyophyllum decastes i and transcriptomic analysis throughout the process |
| topic | functional genomics fungi protoplast regeneration RNA-seq |
| url | https://www.mdpi.com/2309-608X/10/12/886 |
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