一种有效的双孢蘑菇原生质体制备及瞬时转化体系

An effective protoplast preparation and transient transformation system of Agaricus bisporus

原生质体的制备与再生是双孢蘑菇进行遗传转化的基础,通过研究得到制备双孢蘑菇原生质体的最佳条件是:取培养15d的菌丝振荡培养7d,溶壁酶浓度为1mg/mL的0.6mol/L KCl酶解液、温度30℃、45r/min条件下摇培10h,经过富集精制后的原生质体(4×106/mL)可进行瞬时转化。利用Ab-eGFP进行转化,在20min、24h、48h后可观察到GFP荧光,并且在24h和48h可恢复细胞壁增殖,瞬时转化后亦可复壁增殖。研究结果为双孢蘑菇原生质体的稳定遗传转化及后续利用原生质体建立CRISPR-Cas9基因组编辑系统等提供一定的理论依据。

The preparation and regeneration of protoplasts are the basis of genetic transformation of Agaricus bisporus.In this study,an efficient system for protoplast preparation is established through shake culture of 15 days old hyphae for 7 days in liquid medium.Using 1 mg/mL dissolving cell wall enzyme 0.6 mol/L KCl solution resulted in transformation of 4×106/mL protoplasts in 10 h digestion under the temperature of 30°C and shake of 45 r/min.The quality of the isolated protoplasts was verified using vector with Ab-eGFP label.Transient transformation of protoplasts was carried out using PEG4000,and GFP fluorescence was observed in 20 min,24 h and 48 h.Restoration of cell wall was observed in 24 hours and 48 hours.Our result provides a basis for stable genetic transformation of Agaricus bisporus and the subsequent establishment of the crispr-cas9 genome editing system.