一种产黄青霉突变体库的简单快速构建方法

A Simple and Rapid Method for Pennicillium chrysogenum Mutant Library Construction

产黄青霉(Pennicillium chrysogenum)是重要的工业丝状真菌,为更好地提高青霉素产量,了解青霉素合成的调控途径及相关基因的功能,突变体库的构建是一种有效途径。本研究以PCR扩增得到的T-DNA片段为外源DNA,通过PEG介导转化至产黄青霉原生质体中,成功地将含有外源博来霉素抗性基因(ble)和绿色荧光蛋白基因(gfp)的T-DNA插入到产黄青霉基因组中,构建了产黄青霉突变体库,并实现了ble基因和gfp基因在产黄青霉中的表达。该方法不依赖于农杆菌介导,无需构建二元表达载体,只需一步PCR即可实现外源基因的制备,简单快速,也为研究其它真菌基因功能和突变体库构建提供参考。

Pennicillium chrysogenum is an important industrial filamentous fungus for penicillin producing. To increase penicillin production and understand regulation of the penicillin biosynthesis and the corresponding genes function, it is an effective method to construct mutant libraries. In this study, the PCR cloned T-DNA fragments were transformed into P. chrysogenum protoplast by PEG mediated method. The T-DNA fragments containing ble gene and/or gfp gene were integrated into P. chrysogenum genome and the ble gene and/or gfp gene expressed in the transformants. The method does not depend on agrobacterium mediation and it is not necessary to construct the binary expression vector. It is simple and rapid. It provides the basis for other fungi’s gene function and mutant libraries construction.