基于PCR的两步法丝状真菌基因敲除方法

Gene Knockout Method of Filamentous Fungi Based on Two-step PCR Amplification

基因敲除是研究真菌基因功能的重要方法之一,目前,丝状真菌敲除多采用基因两侧的同源臂对基因组的靶标进行同源替换,然后采用抗生素对转化子进行筛选,但这存在着筛选效率低、假阳性率高、操作复杂等问题。本研究以稻瘟菌为研究对象,采取以下步骤对此进行优化:1)构建HPH-GFP(潮霉素-绿色荧光蛋白)融合表达载体; 2)分别用带有接头序列的引物克隆待敲除基因上、下游片段; 3)克隆上、下游片段与HPH-GFP组成嵌合体片段; 4)嵌合体片段转化真菌原生质体; 5)对敲除突变体进行筛选。结果表明,该法操作简便、高效,除了用潮霉素HPH作为筛选标记外,还可用GFP作为荧光筛选标记,极大提高了筛选效率,可用作丝状真菌基因功能研究。

Gene knockout is one of the important methods to study gene function.Usually,gene flanking fragments are used to substitute its genomic homogeneous sequences by using antibiotic as selected marker,but there are still many problems to be solved,like screening inefficiency,high false positive,and complicated operation.In this study,we optimized the knock out techniques using a filamentous fungi Magnaporthe oryzae as an example.The gene knockout strategy includes:1)HPH-GFP fusion expression vector construction.2)The specific gene flanking sequences amplification;3)The chimera fragments composition;4)Fungi protoplast transformation.5)Transformants identification.The results demonstrated that the method improves the mutant identification efficiently by using hygromycin and GFP as screen markers,which could be salutary for gene function investigation of filamentous fungi.