黑曲霉缺失突变体菌株ΔsodC的构建

Construction of the Deletion Mutant ΔsodC

为探究黑曲霉中的超氧化物歧化酶编码基因sodC在其抗氧化代谢方面所起的作用,构建得到黑曲霉缺失突变体菌株ΔsodC。根据同源重组的技术原理,以黑曲霉基因组DNA为模板,扩增获得目的基因sodC的上下游侧翼片段,利用重叠序列对上下游片段进行PCR连接。将该片段与质粒pC3进行连接,并将其转入DH5α感受态细胞,通过蓝白斑筛选及PCR鉴定,得到已构建成功的重组质粒。通过CaCl_2/PEG转化法将重组质粒转入黑曲霉原生质体。通过多步骤的筛选及基因组DNA序列的检测,得到所需突变体菌株ΔsodC以备后续研究使用。

In order to investigate the role in terms of the gene sodC in the antioxidant metabolism in Aspergillus niger,the deletion mutant ΔsodC was constructed. According to the principal of homologous recombination,the upstream and downstream flanking fragments of the gene sodC were amplified by using the genomic DNA of Aspergillus niger as template. Then the upstream and downstream flanking fragments were connected with PCR by using overlapping sequences. The fragment ABCD was connected with plasmid pC3 and transferred into the DH5α competent cells. The recombinant plasmid pC3-ABCD was constructed successfully by blue spot screening and PCR identification.The recombinant plasmid was transferred into protoplast of Aspergillus niger by CaCl2/PEG transformation method. Through multi-step screening and the detection of genomic DNA sequences,we could get the deletion mutant ΔsodC,which could be used for the future research.