Li Xiaobin, Qin Ying, Kong Yufei, Karunarathna Samantha Chandranath, Liang Yunjiang, Xu Jize
College of Agriculture, Yanbian University, Yanji 133002, China.
College of Agriculture, Jilin Agricultural Science and Technology University, Jilin 132000, China.
J Fungi (Basel). 2024 Dec 21;10(12):886. doi: 10.3390/jof10120886.
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 . The results indicated an optimal protoplast yield of 5.475 × 10 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 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.
原生质体是真菌遗传操作和功能基因组学研究的重要工具。本研究系统优化了原生质体制备条件，并检测了整个制备和再生过程中的转录变化，以阐明真菌中原生质体形成和再生的分子机制。结果表明，在真菌培养10天、消化时间2.25小时、酶浓度2%、消化温度28℃的条件下，原生质体产量最佳，为5.475×10个细胞/毫升。添加菌丝提取物的Z5培养基再生率高达2.86。RNA测序分析显示，原生质体形成过程中有2432个差异表达基因（DEG），再生过程中有5825个DEG。酪蛋白激酶I、细胞色素P450（CYP52）和氧化还原调节输入受体（PEX5）在原生质体阶段显著上调，而β-1,3-葡聚糖合酶（SKN1）、几丁质合酶（CHS2）、疏水蛋白-1和疏水蛋白-2在原生质体再生阶段显著上调。这些发现为原生质体的高效制备和再生提供了参考，并为真菌原生质体形成和细胞壁再生的分子机制提供了新的见解。
