生物技术通报Issue(3)：171-177,7.DOI:10.13560/j.cnki.biotech.bull.1985.2015.04.025
黑曲霉柠檬酸工业菌株原生质体制备与转化
Preparation of Protoplast for Efficient DNA Transformation of Citric Acid Hyper-producingAspergillus niger Industrial Strain
摘要
Abstract
Aspergillus niger is the major industrial strain for citric acid production. In spite of many successes of modern molecular biology approaches in engineering laboratory or protein-producing strains ofA. niger, there is few positive report on its application for citric acid industrial strains mainly due to the hard-to-transform nature of these strains. In this study, the protoplast-PEG mediated genetic transformation system for citric acid industrial strain was extensively studied, suggesting an optimized protocol for protoplast preparation, regeneration and DNA transformation. The concentration of protoplasts reached up to 106 /mL by lysing younger mycelia for 2.5 h after 48 h incubation of a proper amount of conidia spores in enrichment medium. The optimal lysing enzyme mixtures comprised of 1.5% lysing enzyme, 0.5% snail enzyme and 0.2% lysozyme. Concentration of protoplast influenced the protoplast-PEG mediated transformation efficiency, which reached the maximal when the concentration of protoplast was higher than 106/mL. The genetic transformation system established in this study should pave the way to molecular biology study of the citric acid hyper-producing strains, for further understanding its acid-tolerant physiology and for rational design of the industrial strain for further improvement of the citric acid production process as well as creation of new organic acid-producing cell factories.关键词
黑曲霉/柠檬酸/遗传操作系统/原生质体/DNA转化Key words
Aspergillus niger/citric acid/genetic manipulation system/protoplast/DNA transformation引用本文复制引用
张晓立,郑小梅,满云,罗虎,于建东,郑平,刘浩,孙际宾..黑曲霉柠檬酸工业菌株原生质体制备与转化[J].生物技术通报,2015,(3):171-177,7.基金项目
国家“863”计划（2013AA020302），国家自然科学基金面上项目 （）
