原生质体法介导真菌遗传转化的研究进展

Research progress in transformation of fungi mediated by protoplasts

原生质体法介导真菌遗传转化体系是实现大规模随机、定点突变和菌种改良的方法之一,为研究真菌致病性和侵染机制奠定了基础。原生质体法构建转化体系的步骤包括原生质体的制备、转化与再生,其中关键是原生质体的制备,通常采用酶解法破除细胞壁获得原生质体,其获得率直接影响转化效率。转化通常借助于聚乙二醇(PEG)介导、电击或限制性内切酶法完成,其中PEG介导的原生质体转化法较传统,技术较成熟;电击法操作较简单;限制性内切酶(REMI)介导的整合转化,因其转化效率较高,可产生大量稳定的转化子,正被越来越多地应用于丝状真菌的遗传转化。

The use of protoplast-mediated genetic transformation system is one of the means to achieve large-scale randomized and site-specific mutagenesis as well as strain improvement in fungi.It lays a foundation for studying fungal pathogenicity and infection mechanism.The procedure of constructing aprotoplast-mediated transformation system involves protoplast preparation,transformation and regeneration.Among them,protoplast preparation is the key step,which is usually obtained by enzyme digestion-based cell wall removal.Protoplast production rate directly affects transformation efficiency.Transformation is usually mediated by polyethylene glycol（PEG）,electric shock or restriction endonuclease digestion;PEG-mediated protoplast transformation is a relatively traditional and mature technique.The electric shock method is featured by simple operation.Because of the higher transformation efficiency which can produce a large number of stable transformants,restriction enzyme mediated integration（REMI）method is increasingly used in the genetic transformation of filamentous fungi.