灭蚊真菌贵阳腐霉原生质体的制备和再生研究

Preparation and Regeneration of Protoplasts of Pythium guiyangense Su,A Fungal Pathogen of Mosquitoes

目的:建立贵阳腐霉原生质体的制备和再生系统,为该菌的基因工程和细胞工程改良提供适宜的真菌状态。方法:对影响原生质体形成的各因素,包括菌龄、消化酶的种类和比例、消化时间以及渗透压稳定剂的成分和浓度进行实验比较。结果:以1%纤维素酶与1%溶壁酶1:1组合联合脱壁,0.6mol/L的甘露醇作渗透压稳定剂,酶解采用KPYG2培养基培养52～54h的贵阳腐霉菌菌丝,消化5h所获得的原生质体数最多,原生质体浓度可达到6.48×106个/ml;在0.6mol/L甘露醇作渗透压稳定剂的KPYG2培养基上,原生质体再生率为0.043%。结论:本实验所总结的原生质体制备方法可以获得在基因转化或细胞突变诱导所需的原生质体浓度。

Objective： To provide a suitable fungal material for protoplast-mediated genetic transformation and mutation induction of Pythium guiyangense Su. Methods： Conditions for the fungal protoplast preparation and regeneration including mycelium incubation time, various compounding of enzymes and osmotic stabilizers were examined. Results： Under the conditions as following： mixture of 1% lywallzyme and 1% cellulose （ 1：1 ） as digestive enzyme solution; 0.6mol/L D-Mannitol as osmotic stabilizer, and 5h, of digestive time the obtained protoplast amount reached 6.48 × 10^6 each milliliter. A regeneration rate of 0.043% was obtained. Conclusion： A satisfied concentration of P. guiyangense protoplasts can be achieved with the preparation system developed in this research.