一种两步基因同源重组敲除酵母靶标蛋白基因的方法

目的:建立一种在代谢工程改造的毕赤酵母菌中高效敲除靶标蛋白基因的方法。方法:构建敲除载体,以粒细胞-巨噬细胞集落刺激因子(GM-CSF)基因为靶基因,以尿嘧啶合成关键酶URA3基因为营养缺陷型筛选标记,第一步重组利用ura3正筛选获得敲除载体在酵母染色体中的定点整合,第二步通过5-氟乳清酸(5-FOA)筛选到表型为ura3-的克隆,ura3基因被剔除的同时,目的基因GM-CSF也随之丢失,实现第二次重组;利用基因组PCR和蛋白电泳进行鉴定。结果:通过两步基因同源重组,敲除了毕赤酵母GJK01的报告蛋白GM-CSF的编码基因388 bp,PCR结果显示该基因已完全丢失,SDS-PAGE分析无GM-CSF表达。结论:仅通过2周时间的筛选、鉴定,在毕赤酵母GJK01中敲除了报告蛋白GM-CSF的编码基因,突变菌株的阳性率达到50%,最终建立了以URA3为筛选标记的两步基因同源重组敲除目的基因的方法。

A High Efficient Method to Knockout Target Gene by Two-Step Homologous Recombination in Pichia pastoris

Objective： To construct a high efficient method to knockout target protein gene by two-step homologous recombination in Pichia pastoris.Methods： The vector of knockouting target protein gene GM-CSF（granulo-cyte-macrophage colony-stimulating factor） in yeast was constructed firstly.First homologous recombinant step is using ura3 gene encoding orotidine-5＇-phosphate decarboxylase as seletable marker to screen positive colonies.Second homologous recombinant step is using 5-FOA（5-uoroorotic acid） gene to screen the mutant strain（△ura3,△GM-CSF）.Results： Using the two-step homologous recombination,the targert protein gene GM-CSF was deleted in yeast strain GJK01.Conclusion： It is a high efficient method to knockout target gene by two-step homologous recombination in yeast.