草菇原生质体制备、再生条件的响应面法优化及诱变效应

Optimizing Conditions on Protoplasts Preparation,Regeneration by Response Surface Methodology and Mutagenesis of Volvariella volvacea

为了提高草菇原生质体在筛选耐低温突变筛选的诱变效率,建立基于草菇原生质体制备产量及再生率的响应面模型,优化草菇原生质体制备体系。本研究以草菇V23为材料,利用Design-Expert 8.0.6软件进行二次回归分析对参数菌龄、酶的浓度、酶解时间、酶解温度等进行了系统的优化研究。结果表明,溶壁酶的浓度为1.71%,酶解温度为31.4℃,菌丝体菌龄为3.42 d,酶解时间为3.21 h,草菇原生质体产量最高,达到2.57×107个/m L;用1.84%的溶壁酶在31.2℃条件下对菌龄为3.68 d的菌丝体酶解3.38 h,原生质体再生率最高,为14.1%。并利用紫外诱变和化学诱变技术对草菇原生质体进行了耐低温诱变初探,筛选获得5株抗冻能力提高的突变菌株。草菇V23原生质体产量和再生率的优化条件的建立,为提高草菇在遗传转化、基因组重排、基因编辑技术等后续分子遗传学操作实验打下了良好的基础。

In order to improve the mutagenesis efficiency of Volvariella volvacea protoplast on low temperature resistant strain selecting, we established response surface model for optimizing Volvariella volvacea protoplast preparation and regeneration conditions, which including mycelial age, enzyme concentration, enzymolysis time and enzymolysis temperature. Mushroom strain was V23, and the parameters were analyzed by Design-Expert 8.0.6 software. The results showd that the yield of protoplast arrived at the highest level of 2.57 ×10~7/m L in the condition of lyase concentration 1.71%, enzymolysis time 3.21 h, mycelium age 3.42 days and enzymolysis temperature 31.4℃. Protoplast regeneration rate with highest level of 14.1% was found in the condition of lyase concentration 1.84%, enzymolysis time 3.38 h, mycelium age 3.68 days and enzymolysis temperature 31.2℃. The ultraviolet mutagenesis and chemical mutagenesis were used to test the protoplast low temperature resestant activities, and five low temperature resistant strains were obtained. The establishing optimized protoplast preparation and regeneration system of Volvariella volvacea, could provide the technical support of protoplasts transformation,genome shuffling and genome targeted editing for Volvariella volvacea.