ZOU Zhangyi,DENG Baiwan.Optimization of Protoplast Preparation and Regeneration Conditions of Agaricus blazei[J].Northern Horticulture,2020,44(03):128-137.[doi:10.11937/bfyy.20192866]
姬松茸原生质体制备及再生条件优化
- Title:
- Optimization of Protoplast Preparation and Regeneration Conditions of Agaricus blazei
- Keywords:
- Agaricus blazei; protoplast yield; regeneration rate; single factor test; orthogonal experiment
- 文献标志码:
- A
- 摘要:
- 以姬松茸JS01为试材，通过单因素试验和正交实验，利用SPSS 22.0软件进行分析，对姬松茸原生质体的制备及再生条件进行优化，以提高姬松茸原生质体产量和再生率。首次通过在再生培养基中添加细胞壁前体物质及营养因子，进一步提高姬松茸原生质体的再生率。结果表明：原生质体最佳制备条件为菌龄7 d，以0.6 mol·L-1 KCl作渗透压稳定剂，溶壁酶浓度1.5%，酶解温度30 ℃，酶解时间4.0 h，pH 6.0，在该条件下原生质体产量高达1.97×107个·mL-1；菌龄6 d，以0.6 mol·L-1 MgSO4作渗透压稳定剂，溶壁酶浓度2.0%，酶解温度30 ℃，酶解时间3.0 h，pH 6.5，再生培养基为GM时，原生质体的再生率最高，为1.12%。再生培养基中添加纤维二糖和维生素B1后，再生率为1.17%，较优化结果提高了4.46%。该研究为姬松茸在菌种提纯复壮、原生质体诱变、原生质体融合育种、原生质体基因组重排等遗传学研究提供参考依据。
- Abstract:
- In order to improve the production and regeneration rate of Agaricus blazei Murrill,this experiment used A.blazei Murrill JS01 as the material,through single factor test and orthogonal experiment,using SPSS 22.0 software to analyze,optimize the preparation and regeneration of Agaricus blazei Murrill.This experiment was the first time to increase the regeneration rate of Agaricus blazei Murrill protoplast by adding cell wall precursors and nutritional factors to the regeneration medium.The results showed that the optimal preparation conditions of protoplast were as followed,fungal age 7 days,0.6 mol·L-1 KCl as osmotic stabilizer,lysozyme enzyme concentration 1.5%,enzymatic temperature 30 ℃,enzymatic hydrolysis time 4.0 hours,and pH 6.0.Under this condition,the protoplast yield was as high as 1.97×107 ·mL-1;the fungal age was 6 days,0.6 mol·L-1 MgSO4 was used as the osmotic stabilizer,the lysozyme enzyme concentration was 2.0%,and the enzymatic temperature was 30 ℃.When the time was 3.0 hours,pH 6.5,the regeneration medium was GM,the regeneration rate of protoplast was the highest,1.12%.After adding cellobiose and vitamin B1 to the regeneration medium,the regeneration rate was 1.17%,which was 4.46% better than the optimized result.This study provided a theoretical basis for genetic research on A.blazei Murrill in purification and rejuvenation of fungal,protoplast mutagenesis,protoplast fusion breeding,and protoplast genome rearrangement.
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备注/Memo
第一作者简介：邹彰毅（1994-），男，硕士研究生，研究方向为微生物资源保育与应用开发。E-mail：260332661@qq.com.责任作者：邓百万（1963-），男，硕士，教授，现主要从事微生物资源保育与应用开发等研究工作。E-mail：2210309868@qq.com.基金项目：陕西省重点研发计划资助项目(2017ZDXM-NY-001)。收稿日期：2019-09-17