3.11 Analysis of relative expression levels of genes
Based on the measurements of Monascus growth and development through different experiments using various nitrogen sources, it can be concluded that the addition of glutamine as a nitrogen source in the medium does not affect the growth and development of monascus. However, when NaNO3 is used as the nitrogen source, knockdown and overexpression of the AreA gene have a significant impact on monascus growth and development. Therefore, we analyzed the expression of four key genes involved in red currant pigment biosynthesis (MpPKS5, mppG, mppD, mppE ) and four genes related to growth and reproduction (VeA, VosA, LaeA, GprD ) in Aspergillus rubra’s original strain C100 as well as in the knockout strain ΔMareA and overexpression strain OE-MareA .
There are 17 key genes on the MPs synthesis gene cluster. Among them,MpPKS5 and mppD are structural genes in the erythro-pigment polyketide synthesis pathway, playing crucial roles in the process of pigment synthesis. MppD is a homolog ofMpPKS5 and has been suggested to have a specialized but undetermined auxiliary role to MpPKS5. MppE acts as a reductase in the polyketide pathway, controlling the biosynthesis of yellow pigment. MppG is closely involved in synthesizing reddish currant orange and red pigments(Chen et al., 2017).
As shown in Figures 3-17, the relative expression of MpPKS5 and mppD genes in the knockout strain ΔMareA was decreased under control conditions without the addition of nitrogen source. The expression of mppE gene was significantly up-regulated. The relative expression levels of MpPKS5, mppG, mppD and mppE genes in the OE-MareA were significantly down-regulated. These show that the quantity of key gene expression MareA gene of red kojic rice fermentation have played an important role in the pigment metabolism. In solid fermentation medium supplemented with Gln, the expression of MpPKS5 gene in ΔMareA was essentially unchanged, whereas the relative expression of mppG, mppD and mppE genes all decreased. The relative expression levels of MpPKS5, mppG, mppD and mppE genes in OE-MareA were up-regulated to varying degrees. After addition of NaNO3, the expression of MpPKS5 gene in ΔMareA was close to that in C100, while the relative expression of mppG, mppD and mppE genes was significantly up-regulated. In OE-MareA , the expression of MpPKS5 and mppE genes was down-regulated, while that of mppG and mppD genes was up-regulated. This indicates that the loss ofMareA gene resulted in increased expression of pigmentation genes after addition of NaNO3, and that overexpression of these genes resulted in a significant reduction in yellow pigment accumulation.
In summary, the MareA gene affects the expression of pigment genes. When Gln was added, the deletion of MareA gene suppressed the expression of key pigmentation genes, while overexpression promoted it. When the nitrogen source NaNO3 was added, the deletion of MareA gene promoted it more obviously compared to overexpression.