一种高效、定向构建产紫杉醇真菌营养缺陷型的方法

Efficient and targeted method for constructing auxotrophs of paclitaxol-producing fungus

为定向获得某种特定的、遗传特性稳定的营养缺陷型菌株,解决产紫杉醇真菌PM6基因改造过程中遗传筛选标记不足的问题,通过基因敲除技术建立高效构建特定营养缺陷型菌株的方法,并将该方法经过简化,作为综合实验"基因敲除法构建产紫杉醇真菌营养缺陷型菌株"应用于本科生实验教学。以构建产紫杉醇真菌腺嘌呤营养缺陷型菌株为例,通过基因同源重组方法定向敲除PM6菌株腺嘌呤合成途径关键基因ADE,利用分子生物学方法和菌落形态分析筛选鉴定缺陷型突变株。通过构建营养缺陷型菌株,为丝状真菌的转化提供了新的筛选标记。使学生系统地了解和掌握了基因定向敲除技术,提高了本科生综合实践能力和科研素养,取得了良好的教学成果。

In order to obtain a specific auxotrophic strain with stable genetic characteristics and solve the problem of insufficient genetic selection markers during the genetic modification of the paclitaxel-producing fungus PM6, a method for efficient construction of specific auxotrophic strains is established through gene knockout technology, which is simplified and used as a comprehensive experiment "Gene knockout method to construct auxotrophic strains of paclitaxol-producing fungus" and applied to experimental teaching of undergraduates. Taking the construction of a paclitaxol-producing fungus adenine auxotrophic strain as an example, this paper introduces the targeted knockout of the key gene of adenine synthesis pathway of PM6 strain ADE through gene homologous recombination method, and uses molecular biology methods and colony morphology analysis to screen and identify defective mutant strains. By constructing auxotrophic strains, new screening markers are provided for the transformation of filamentous fungi, and applied to the experimental teaching of undergraduates, so that undergraduates can systematically understand and master gene-directed knockout technology, and improve their comprehensive practice ability and scientific research literacy, which has achieved good teaching results.