<div>3.11 Analysis of relative expression levels of genes</div><div>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 NaNO<sub>3</sub> is used as the nitrogen source, knockdown and
overexpression of the <i>AreA</i> 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
(<i>MpPKS5, mppG, mppD, mppE</i> ) and four genes related to growth and
reproduction (<i>VeA, VosA, LaeA, GprD</i> ) in Aspergillus rubra’s
original strain C100 as well as in the knockout strain Δ<i>MareA</i> and
overexpression strain OE<i>-MareA</i> .</div><div>There are 17 key genes on the MPs synthesis gene cluster. Among them,<i>MpPKS5</i> and <i>mppD</i> are structural genes in the
erythro-pigment polyketide synthesis pathway, playing crucial roles in
the process of pigment synthesis. <i>MppD</i> is a homolog of<i>MpPKS5</i> and has been suggested to have a specialized but
undetermined auxiliary role to <i>MpPKS5.</i> <i>MppE</i> acts as a
reductase in the polyketide pathway, controlling the biosynthesis of
yellow pigment. <i>MppG</i> is closely involved in synthesizing reddish
currant orange and red pigments(Chen et al., 2017).</div><div>As shown in Figures 3-17, the relative expression of MpPKS5 and mppD
genes in the knockout strain Δ<i>MareA</i> 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-<i>MareA</i> were
significantly down-regulated. These show that the quantity of key gene
expression <i>MareA</i> 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
Δ<i>MareA</i> 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-<i>MareA</i> were
up-regulated to varying degrees. After addition of
NaNO<sub>3</sub>, the expression of MpPKS5 gene in Δ<i>MareA</i> was close to that in C100, while the relative expression of mppG, mppD
and mppE genes was significantly up-regulated. In OE-<i>MareA</i> , 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 of<i>MareA</i> gene resulted in increased expression of pigmentation genes
after addition of NaNO<sub>3</sub>, and that overexpression of
these genes resulted in a significant reduction in yellow pigment
accumulation.</div><div>In summary, the <i>MareA</i> gene affects the expression of pigment
genes. When Gln was added, the deletion of <i>MareA</i> gene suppressed
the expression of key pigmentation genes, while overexpression promoted
it. When the nitrogen source NaNO<sub>3</sub> was added, the
deletion of <i>MareA</i> gene promoted it more obviously compared to
overexpression.</div>