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.