基于响应面法对香菇原生质体制备条件的优化

Optimization of Lentinula edodes protoplast producing conditions based on response surface methodology

香菇Lentinula edodes是我国食用菌年产量最大的单品,优化香菇原生质体的制备条件,获得高质量、高活力的原生质体可以为香菇育种和遗传多样性分析提供技术保障。本研究利用单因素试验对稳渗剂浓度、酶解液类型、酶解时间、酶解温度4个因素的最优作用条件和相关性进行检验和筛选;依据单因素试验的结果,对酶解时间、酶解温度、酶解液种类进行响应面法3因素3水平试验优化分析。单因素试验结果表明,使用2%的溶壁酶+蜗牛酶+纤维素酶混合酶制剂为酶解液、0.6mol/L甘露醇为稳渗剂的效果显著高于其他处理(P<0.05)。经Box-Behnken设计得出香菇原生质体最佳制备条件为酶解时间3.25h,酶解液浓度1.7%,酶解温度30.49℃,获得原生质体产量为14.02×10^(6)CFU/mL,与理论产量(13.862×10^(6)CFU/mL)偏差小,可为后续原生质体融合育种和多样性分析提供重要基础。

Lentinula edodes is the highest-yielding edible mushroom grown in China.Optimizing the protoplast preparation conditions to generate high quality and vitality of the fungal protoplasts is critical for breeding and genetic diversity analysis of L.edodes.The optimal action conditions and correlations of four factors,concentration of osmolymer,enzymatic type,enzymolysis duration and enzymolysis temperature,were tested and screened by single factor test.According to the results of single factor test,the duration and temperature of enzymolysis and enzyme solution were investigated by three factors and three levels of response surface method.The single factor test shows that using mixed enzyme preparation of 2%lysozyme+snail enzyme+cellulase with 0.6 mol/L mannitol as osmotic stabilizer for synergistically multiple enzyme enzymolysis was significantly effective as compared with other treatments(P<0.05).Through Box-Behnken design,the optimal preparation conditions of L.edodes protoplasts were as follows:enzymolysis duration of 3.25 h,enzymolysis concentration at 1.7%,and enzymolysis temperature at 30.5°C.Under these conditions the yield of protoplasts was 14.02×10^(6)CFU/mL,consistent with the theoretical yield(13.862×10^(6)CFU/mL).The results provide a key technological guidance for further protoplast fused breeding and genetic diversity analysis of L.edodes.