巨大侧耳原生质体制备条件的优化

Optimization of protoplast preparation conditions of Pleurotus giganteus

为优化巨大侧耳原生质体的制备条件,该研究以两株不同温型的巨大侧耳菌株PG46和PG79为材料,采用单因素和正交试验方法对原生质体制备的菌丝体菌龄、稳渗剂种类、溶壁酶浓度、酶解温度和酶解时间进行研究。结果表明:(1)在单因素实验中,巨大侧耳原生质体制备的适宜条件为菌龄5 d,溶壁酶浓度2.5%,0.6 mol·L^(-1)甘露醇,32℃(PG46)或27~35℃(PG79)酶解4 h。(2)正交试验验证并优化了单因素实验结果,组合2(菌龄5 d,溶壁酶浓度2.5%,0.6 mol·L^(-1)的甘露醇,32℃酶解4 h)可同时作为PG46和PG79原生质体制备的最适条件,原生质体产量分别为11.22×10^(6)CFU·mL^(-1)和7.28×10^(6)CFU·mL^(-1)。(3)F-test检验中,各因素对原生质体制备的影响程度依次为菌龄>溶壁酶浓度>酶解温度>酶解时间(PG46),菌龄>酶解时间>酶解温度>溶壁酶浓度(PG79)。综上所述,两株不同温型巨大侧耳菌株的原生质体制备条件基本一致,菌龄对两菌株原生质体得率的影响程度最显著。该研究结果可为后续巨大侧耳的杂交育种、遗传转化、全基因组测序等工作奠定基础,进一步推动巨大侧耳分子遗传学的发展。

In order to obtain the optimal conditions for the protoplast preparation of Pleurotus giganteus,two strains,PG46 and PG79,with different temperature types,were selected as materials to study the effects of five factors(mycelial age,osmotic stabilizer type,lywallzyme concentration,enzymatic hydrolysis duration and enzymatic hydrolysis temperature)based on single factor and orthogonal experimental methods.The results were as follows:(1)In the single factor test,the optimal conditions for the protoplast preparation of P.giganteus were mycelial culture for 5 d,using 2.5%lywallzyme with 0.6 mol·L^(-1)mannitol,incubated for 4 h at 32℃(PG46)or 27-35℃(PG79).(2)Orthogonal experiment verified and optimized the single factor test results.Combination 2(mycelial age 5 d,lywallzyme concentration 2.5%,0.6 mol·L^(-1)mannitol,incubated for 4 h at 32℃)could be used as the optimal condition for the protoplast preparation of PG46 and PG79 at the same time,and the protoplast yields were 11.22×10^(6)CFU·mL^(-1)and 7.28×10^(6)CFU·mL^(-1),respectively.(3)For F-test,the influence degree of various factor on the protoplast preparation were mycelial age>lywallzyme concentration>enzymatic hydrolysis temperature>enzymatic hydrolysis duration(PG46),and mycelial age>enzymatic hydrolysis duration>enzymatic hydrolysis temperature>lywallzyme concentration(PG79),respectively.In conclusion,the protoplast preparation conditions of the two P.giganteus strains with different temperature types were basically the same,and the effects of mycelial age on the protoplasts yield of the two strains were the most significant.The results can lay a foundation for further cross-breeding,genetic transformation,whole genome sequencing and promote the molecular genetics development of P.giganteus.