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.