黄曲霉菌原生质体的制备和再生技术优化

Technology and Optimization of Preparation and Regeneration of Protoplasts from Aspergillus flavus

黄曲霉菌的遗传转化是研究黄曲霉菌致病相关功能基因的前提和基础,而原生质体是研究和建立真菌遗传转化系统的重要工具。本文分别以黄曲霉孢子和菌丝为材料,研究不同条件下黄曲霉原生质体的形成和再生,结果表明,黄曲霉孢子在酶液浓度为纤维素酶∶蜗牛酶∶溶壁酶=1.5%∶1.5%∶1.5%,30℃酶解3 h,原生质体制备率高达97.3%,再生率达89.2%;黄曲霉菌丝在菌龄为42 h,酶液浓度为纤维素酶∶蜗牛酶∶溶壁酶=1.5%∶1.5%∶1.5%,30℃酶解1 h,可获得最高原生质体产量为2.0×106个/m L,再生培养基中以1 mol/L蔗糖作为渗透压稳定剂时,原生质体再生率达5.5%。故本实验条件下,黄曲霉孢子原生质体的形成和再生优于菌丝。

The Genetic transformation of Aspergillus flavus is the premise and foundation of the study of related functional genes of this pathogenic fungus. And the protoplasts is an important approach to study and establish the fungal genetic transformation system. The preparation and regeneration of protoplasts of A. flavus spores as well as mycelia were investigated in the paper. The results showed that the optimum condition for protoplasts preparation from A. flavus spores was as follow,cellulase enzyme： Snail enzymes： lywallzyme = 1. 5% ∶ 1. 5% ∶ 1. 5%,30 ℃ water bath 3 hours,and the formation rate of protoplasts was 98. 5% and the regeneration rate was 89. 2%. The maximum protoplasts yield from mycelia was 2. 0 × 10^6/ m L when the thallus cultured for 42 h were hydrolyzed at 30 ℃ for 1h with the combination of 1. 5% lywallzyme,1. 5% snailase and 1. 5% cellulose. The maximum regeneration frequency of protoplasts was 5. 5 % when 1mo L / L sucrose was used as osmotic pressure stabilizer. The results indicated that A. flavus spores were more suitable for preparing protoplasts than mycelia under the experimental conditions in this paper.