Materials and Methods
2.1 Strains, media and culture conditions
Monascus ruber C100: a gift from Prof. Mianhua Chen, the
original strain for gene knockout and overexpression; Escherichia
coli DH5α: kept in our lab for plasmid transformation;Agrobacterium tumefaciens EHA105: Preserved in this laboratory
for use in transformation; p59 plasmid: kept in this lab for the
construction of knockout plasmid vectors; p1301 plasmid: kept in our lab
for the construction of overexpression plasmid vectors.
Inoculate the C100 with Z-shaped wavy lines onto the inactivated MEA
slant, and incubate it at a constant temperature of 30℃ for about 7
days. Store it in the refrigerator at 4℃ and reactivate every six
months.
2.2. Extraction of C100 genome
Genomic DNA was extracted as described by Yu et al(Yu et al., 2004)with
some modifications. Weigh the mycelium into a centrifuge tube, add 1mL
of a 5% CTAB solution and 20μL of β-mercaptoethanol, then use a crusher
to crush it for 1 minute. After crushing, place it in a 65℃ incubator
for 30 minutes to maintain warmth. Mix well by adding an equal volume of
chloroform, followed by centrifugation at 10,000rpm/min at 4℃ for 5
minutes. Take the supernatant and add isopropanol (0.6 times the volume)
and NaAC (0.1 times the volume). Leave it at -20℃ for 2 hours, then wash
with fresh aliquots of ethanol (75%) for a duration of 5 minutes to
remove the supernatant. Remove ethanol after resting for another minute
using fresh aliquots of ethanol (75%). Centrifuge again at -20 ℃ with
NaAC present during stationary phase lasting two hours at a speed of
10000 rpm/min followed by removal of supernatant. Finally, add TE
buffer (50μL) to obtain the genome extract from red currant bacteria.
Extraction of total RNA from monascus was performed using the Fungal RNA
Kit (OMEGA) procedure. The obtained total RNA from Monascus was then
converted into cDNA using the HiFiScript cDNA Synthesis Kit.The reaction
system and procedures are shown in Tables 2-1 and 2-2.
2.3 Construction of knockout and overexpressed recombinant plasmids
Specific primers were designed using C5.761 of the M. purpurus YY-1
genome as the reference sequence. Primers were designed to amplify the
upstream and downstream homology arms of the target gene using the
original strain DNA as a template. Using p59 as the cloning vector, the
recombinant plasmid p59s was obtained after first double digestion of
the vector using PstI and HindIII and ligation according to the
instructions of the seamless cloning kit. Similarly, p59s was double
digested using KpnI and XbaI followed by ligation, resulting in the
final recombinant plasmid p59ss. Primers for the target gene were
designed using C100 cDNA as a template and amplified. The target
fragment and the vector were digested with NocI and AflII, and then
ligated with the DNATAKARA kit to obtain the overexpression vector
p1301s.
2.4 Agrobacterium-mediated transformation
Agrobacterium-mediated transformation was referenced to W. Li et
al.(Li et al., 2023) with fine-tuned corrections. To compare the gene
expression of the MareA gene in the original strain and the knockout and
overexpression strains, total RNA was extracted from both strains and
converted into cDNA. RT-qPCR was performed using the SYBR® Premix Ex
TaqTM II kit from TAKARA for relative quantification. The C100 setup
served as the template in the assay as the calibrator, withβ-actin used
as the internal reference gene. Three replicates were conducted for each
gene. At the end of the reaction, amplification efficiencies of genes
were observed, and when their efficiencies were essentially equal,
comparisons and analyses could be made by comparing and analyzing Ct
values at which gene amplification reached threshold levels. The
relative expression of genes could be analyzed using the
2-ΔΔCt method. The RT-qPCR reaction system is shown in
Tables 2-3, and the RT-qPCR reaction procedure is shown in Tables 2-4.
-ΔΔCt=-[(experimental group gene Ct value - experimental group
internal reference Ct value) - (control group gene Ct value - control
group internal reference Ct value)]
Equation (2-1)
2.5 Observation and Determination of Colonies
Spore suspensions of the original strain C100 , knockout strainΔMareA , and overexpression strain OE-MareA were inoculated
onto PDA, PDA-Gln,
PDA-(NH4)2SO4,
PDA-NaNO3, and PDA-Urea media centers with 5μL each. The
bacterial growth was observed after constant incubation at 30℃ for 7
days.(Liu et al., 2019) .
C100 ,ΔMareA and OE-MareA were cultured on media
supplemented with different nitrogen sources and their diameters were
measured using the criss-cross method.
Cultured C100, ΔMareA , and OE-MareA were incubated
at 30℃ for 7 days. The mycelium and spores on the surface of the medium
were washed off with aseptic water. Then, a 20μL drop was collected on a
slide and observed under a fluorescence inverted microscope (20×) to
assess the growth of the mycelium and spores as well as the intensity of
fluorescence.
C100, ΔMareA , and OE-MareA were cultured on PDA medium
supplemented with different nitrogen sources, respectively. After 7 days
of incubation at 30℃, mycelia and spores were washed off the surface of
the medium with aseptic water, centrifuged at 8,000 rpm for 10 minutes.
After removing the supernatant, they were soaked in 2.5% glutaraldehyde
and fixed overnight in a refrigerator at 4℃. The fixative was then
removed and the mycelium and spores were rinsed three times with PBS
buffer at pH=7.0 for each time interval of 10 minutes. Then the samples
were dehydrated step by step using ethanol concentrations of
30%,50%,70%,80%,90% and finally 100%. Each concentration was
treated for a duration of ten minutes. The processed samples underwent
vacuum freeze-drying process. The samples were glued onto carbon
conductive adhesive, sprayed with gold, and observed under scanning
electron microscope to examine the mycelium and spores.(Zhang et al.,
2019).
2.6 Determination of the color valence of the Mps s(MPs)
Weigh 0.1g of red yeast rice powder in a 10mL centrifuge tube, and add
75% ethanol three times according to the ratio of 4:3:3. Sonicate for
30 minutes each time. Centrifuge at 8000rpm/min for 10 minutes, transfer
the supernatant into a new centrifuge tube, and finally adjust the
volume to 10mL with 75% ethanol. Take 1mL of extracted MPs for
appropriate dilution, add it into a quartz cuvette, and measure its
absorbance at wavelengths of 385nm (yellow pigment),475nm (orange
pigment),505nm (red pigment),and 410nm(total pigment). Use blank
solution containing only75% ethanol to set absorbance between 0.2-0.8
range. Calculate color value using equation(2-2).
Equation (2-2)
Where: S- color value of the sample;
A - Absorbance value of the dilution;
V-80% methanol volume (mL);
m-Dry weight of monascus mycelium (g);
n-dilution times;
2.7 HPLC Determination of MPs
After extracting the pigments, they were diluted to
OD410 ≤ 10 and filtered using a 0.22μm organic filter
membrane before determining the pigment composition through
high-performance liquid chromatography (HPLC). The mobile phases for
pigment detection were as follows: phase A consisted of 0.1% formic
acid in water, while phase B was pure acetonitrile. Both aqueous and
organic phases were filtered with a 0.45μm filter membrane and degassed
by ultrasonic treatment for 30 minutes. HPLC conditions are shown in
Tables 2-5. Pigment detection methods are shown in Tables 2-6.
2.8 Analysis of relative gene expression level
The expression of four key genes involved in MPs biosynthesis
(MpPKS5 , mppG , mppD , mppE ) and four genes
related to growth and reproduction (veA, vosA, laeA, gprD ) were
analyzed in the original strain of M. ruber C100, the knockout
strain ΔMareA , and the overexpression strain OE-MareA .
Primers were designed according to RT-qPCR primer design principles.
cDNAs from C100, ΔMareA , and OE-MareA were used as
templates for RT-qPCR analysis of the above eight related genes. Primers
were designed according to the RT-qPCR primer design principles, as
shown in Tables 2-7.
RESULTS
3.1 Construction and validation of the vectors
Upstream and downstream homology arms were designed at both ends of theMareA gene and inserted into both ends of the hyg gene on
the p59 plasmid, respectively, to achieve gene knockdown. The schematic
diagram illustrating the construction of its knockdown plasmid is shown
in Figure 3-1.
The MareA target gene was inserted between the strong promoter
and terminator of tobacco mosaic virus CaMV 35S on the p1301 plasmid for
the purpose of gene overexpression. The schematic diagram illustrating
the construction of its overexpression plasmid is shown in Figure 3-2.