桑黄原生质体的制备与再生

Research on Protoplast Preparation and Regeneration of Phellinus igniarius

为了优化桑黄(Phellinus igniarius)原生质体制备与再生体系,以桑黄DL101的菌丝体为材料,利用Design-Expert 8.0.6软件进行二次回归分析对参数酶解温度、酶解时间、酶的浓度等进行了系统的优化研究,此外,采用不同的再生培养基对桑黄原生质体的再生进行了研究。获得了制备桑黄原生质体的最佳酶系统、酶解时间以及酶解温度,分别为0.5%崩溃酶+2.0%溶壁酶、酶解3.0 h、酶解温度30℃,此时,原生质体得率最高,为1.49×107个/mL;此外,0.6 mol·L-1硫酸镁作为再生渗透压稳定剂时桑黄原生质体的再生率最高,可达到0.67%。桑黄DL101原生质体制备与再生体系的建立,为提高桑黄在后续分子遗传转化、菌种改良、基因编辑等奠定了良好的基础。

In order to improve Phellinus igniarius protoplaset preparation and regeneration conditions.In this study,DL101 strain mycelium were used as material,and the parameters such as enzymolysis temperature,enzymolysis time and enzyme concentration were analyzed by design-expert 8.0.6 software.Moreover,regeneration of P.igniarius protoplast was performed on different kinds of regeneration medium.The optimum protoplast preparation of P.igniarius was obtained.The yield of protoplast arrived at the highest level of 1.49×107/mL in the condition of driselase 0.5%+lywallzyme 2.0%,enzymolysic time 3.0 h and enzymolysis temperature 30℃.In addition,the protoplast regeneration rate of P.igniarius with highest level of 0.67%was found in 0.6 mol·L-1 MgSO4 stabilizer.The establishing optimized protoplast preparation and regeneration system of P.igniarius could lay the foundation of protoplasts transformation,strain engineering and genome targeted editing for P.igniarius.