Fig.3-2 Construction diagram of MareA gene overexpression vector
The RT-qPCR was performed using the cDNA of C100 and ΔMareA as templates, with qMareA-F and qMareA-R serving as primers. The expression of the MareA gene in both strains was compared through calculation. By comparing the expression levels of the MareA gene in C100 and ΔMareA strains, it was observed that the gene could be expressed normally in the original strain C100, while its expression in the knockout strain ΔMareA approached zero. This suggests that there is almost no transcriptome of the MareA gene in ΔMareA, further confirming successful knockdown of theMareA gene.
The cDNAs of C100 and OE-MareA were used as templates for RT-qPCR, with qMareA-F and qMareA-R serving as primers. The expression of the MareA gene in both strains was compared through calculation. The relative expression level of the MareA gene in the overexpression strain OE-MareA was found to be 3.41 times higher than that in the original strain C100, indicating successful insertion of the MareA gene fragment into the genome of monascus C100 and successful screening of the overexpression transformantOE-MareA . The results are presented in Figure 3-3.