玫烟色棒束孢IfB01菌株原生质体制备与转化条件研究

摘要

In order to increase the yield and transformation efficiency of protoplast from Isaria fumosorosea strain IfB01,an entomopathogenic fungus,and then to lay a foundation for the strain improvement by genetic and cell engineering,the effects of selected parameters( concentration ratio of cell wall-degrading enzyme,en⁃zyme digestion time,temperature,pH value,rotating speed and concentration of osmotic stabilizer) on proto⁃plast preparation of I.fumosorosea strain IfB01,and the effects of different concentrations of PEG4000 on trans⁃formation efficiency of the fungus were examined.The highest protoplast yield of 6.72×106 per milliliter was ob⁃tained from 30 h-old mycelia after treatment with 1% snailase-2% cellulose,osmotic stabilizer of 0.7 mol/L NaCl(pH6.0),8 h of enzymolysis under 30℃,100 r/min.The highest transformation efficiency of 7.05% was obtained under the treatment of 40% PEG4000.It showed that enzyme solution,hydrolysis temperature,time, pH,osmotic pressure,stabilizer,speed and concentration of PEG4000 have great influence on protoplast prepa⁃ration and transformation.%为了提高虫生真菌玫烟色棒束孢IfB01原生质体的产量和转化效率，进而为该菌株的基因工程和细胞工程改良奠定基础，本文研究了不同细胞壁降解酶配比、渗透压稳定剂、酶解温度、时间、pH值和转速等对玫烟色棒束孢IfB01原生质体制备以及不同聚乙二醇4000（ PEG4000）浓度对原生质体转化的影响。结果显示，培养30 h的菌丝用1％蜗牛酶＋2％纤维素酶配比、以0．7 mol／L 的 NaCl 溶液（pH6．0）为渗透压稳定剂，30℃、100 r／min酶解8 h，获得的原生质体产量最高，达6．72×106个／mL；此原生质体在40％的PEG4000作用下可获得最高的转化效率，达7．05％。研究表明酶液、酶解温度、时间、pH值、渗透压、稳定剂、转速和PEG4000的浓度等因素对原生质体的制备和转化有较大影响。