河南农业大学学报2024，Vol.58Issue(4)：592-600,9.DOI:10.16445/j.cnki.1000-2340.20240227.001
黑曲霉糖化酶工业菌株CBS 513.88原生质体制备条件的优化
Optimization of protoplast preparation for an industrial glucoamylase-producing strain Aspergillus niger CBS 513.88
摘要
Abstract
[Objective]The conditions of protoplast preparation were optimized for the industrial glu-coamylase-producing strain Aspergillus niger CBS 513.88,thus facilitating its basic research and mo-lecular modification.[Method]In this study,the protoplast preparation conditions of Aspergillus niger CBS 513.88 were optimized by examining the effects of enzymatic hydrolysis solution composition and proportion,enzymatic hydrolysis conditions,osmotic stabilizer,mycelium growth conditions on the for-mation of protoplasts.The protoplasts transformation efficiency of Aspergillus niger CBS 513.88 was also tested by polyethylene glycol-mediated protoplast transformation method.[Result]For protoplast prepa-ration of Aspergillus niger CBS 513.88,the optimal protoplast preparation material was the mycelium cul-tivated in modified ME medium for 10 hours,and the best enzymolysis condition was carried out at 33 ℃ and 110 r·min-1 for 1.5 hours in a solution(pH 5.8)of 10 g·L-1 Glucanex® and 10 g·L-1 lysozyme,with the optimal osmotic stabilizer of 0.7 mol·L-1KCl+0.27 mol·L-1CaCl2.Under the above conditions,the concentration of protoplast suspension can reach up to 2 × 107 protoplasts·mL-1.Besides,the best protoplasts regeneration medium was ME medium supplemented with 0.7 mol·L-1KCl,whose maximum regeneration rate was 52％.The transformation efficiency was up to 206 trans-formants per μg DNA of plasmid,and the positive rate was 94.4％.[Conclusion]In this study,a short and efficient protoplast-mediated genetic transformation system was established by optimizing the protoplast preparation conditions of Aspergillus niger CBS 513.88.关键词
黑曲霉CBS 513.88/原生质体制备/原生质体再生/原生质体介导转化/遗传转化体系Key words
Aspergillus niger CBS 513.88/protoplasts preparation/protoplasts regeneration/proto-plast-mediated transformation/genetic transformation system分类
生物科学引用本文复制引用
赵君,张震,董飞宇,徐轶凡,陈红歌..黑曲霉糖化酶工业菌株CBS 513.88原生质体制备条件的优化[J].河南农业大学学报,2024,58(4):592-600,9.基金项目
国家自然科学基金项目(32171476) （32171476）
