Preparation and Regeneration of Didymella bryoniae Protoplasts
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摘要:
目的 建立高效的病原菌原生质体制备和再生体系，明确甜瓜蔓枯病菌的致病机理。 方法 采用单因素分析法，利用2种混合酶20 g·L−1 Driselase和8 g·L−1 Lysing enzymes，以不同菌龄、摇培转速、不同酶解时间、温度、渗透压稳定剂的种类及pH、再生培养基为研究条件；对甜瓜蔓枯病菌原生质体的制备及再生体系进行优化。 结果 结果表明：以培养36 h左右的菌丝体为材料，以0.7 mol·L−1 NaCl作为渗透压稳定剂（pH=7），用20 g·L−1 Driselase和8 g·L−1 Lysing enzymes在30 ℃、140 r·min−1条件下酶解4 h，原生质体的产量可达9.65×107个·mL−1；以琼脂含量为0.6%的SR培养基作为再生培养基，甜瓜蔓枯病菌的再生率可达22.53%；原生质体的再生率在存放10 h内下降迅速，10 h后下降速度逐渐平缓。 结论 确定了蔓枯病菌的高效原生质体制备与再生体系，为该菌株遗传转化体系的建立奠定基础。 Abstract:Objective To better understand the pathogenicity of Didymella bryoniae on melons, this study aimed to establish an efficient means of preparing and regenerating the fungal protoplasts. Method Driselase at 20 g·L−1 and a lysing enzyme at 8 g·L−1 were combined and used for the protoplast preparations. A single-factor experiment was conducted to analyze the effects of age of mycelia, time and temperature of enzymatic digestion, rotational speed of culture vessel, type and concentration of osmotic stabilizer, and pH of medium on releasing of the protoplasts. Conditions for the subsequent protoplast regeneration in medium were also optimized. Result The highest yield of protoplasts of 9.65×107 cells·mL−1 was achieved using the mycelia cultured for 36 h to be enzymatically digested in a solution containing NaCl 0.7 mol ·L−1 as the osmotic stabilizer at 30 ℃ and pH 7.0 with 140 r·min−1 constant rotation for 4 h. The protoplast regeneration rate could reach up to 22.53% in a 0.5% agar SR culture medium but declined rapidly in 10 h and then leveled off in storage. Conclusion The protoplasmic preparation and regeneration methods were efficient and could materially aid the establishment and research on the genetic transformation of D. bryoniae. -
Key words:
- Didymella bryoniae /
- protoplast /
- preparation and regeneration
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图 1 菌丝菌龄对原生质体产量的影响
注: 不同的小写字母代表差异显著（P＜0.05），下同
Figure 1. Effect of mycelium age on protoplast release by lysis
Note: Data with different lowercase letters represent significant differences (P＜0.05). Same for the following.
图 2 酶解时间和酶解温度对原生质体产量的影响
注: A: 酶解时间对原生质体产量的影响; B: 酶解温度对原生质体产量的影响
Figure 2. Effects of enzymatic digestion time and temperature on protoplast release by lysis
Note: A: effect of enzymatic digestion time; B: effect of enzymatic digestion temperature.
图 3 渗透压稳定剂对原生质体产量的影响
注: A: 不同稳定剂对原生质体产量的影响; B: NaCl的浓度与原生质体产量的相关性
Figure 3. Effect of osmotic stabilizer on protoplast release by lysis
Note: A: effects of different stabilizers; B: correlation between NaCl concentration and protoplast yield.
图 4 pH和转速对原生质体产量的影响
注: A: pH对原生质体产量的影响; B: 摇培转速与原生质体产量的相关性
Figure 4. Effects of pH and rotational speed on protoplast yield in lysis
Note: A: effect of pH; B: effect of rotational speed.
图 5 不同再生培养基对原生质体再生率的影响
注: A: 不同再生培养基对原生质体再生的影响; B: 上层覆盖培养基含量与原生质体再生的相关性
Figure 5. Effects of medium and agar concentration on protoplast regeneration rate
Note: A: effect of regeneration medium; B: effect of concentration of superstratum medium.
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