| Monascus spp. are the producers of red yeast rice (RYR), also called Hongqu, which have been widely used as food additives, traditional medicine, as well as fermentation starters for many centuries in oriental countries. In recent years, a variety of bioactive metabolites, such as monacolins, gamma aminobutycic acid, pigment and antioxidant (dimerumic acid) et al., have been isolated and identified; hence, biosynthetic pathway of these secondary metabolites attract extensive attention from researchers. Previous studies have demonstrated that the biosynthesis of secondary metabolites in fungi is not only associated with structural genes, but also with different kinds of regulatory genes, and thus researches on regulatory genes are conducive to the explanation of the molecular regulatory mechanism of secondary metabolites biosynthesis in Monascus spp..Regulators of G-protein signaling (RGSs) are a group of proteins which govern the intensity and duration of G-protein signaling by interacting with an activated GTP-G proteinαsubunit (Ga). Functional analysis of regulators of G-protein signaling in many filamentous fungi indicated that they play pivotal roles in the regulation of developmental and secondary metabolic processes including vegetative growth, sporulation, mycotoxin/pigment production and pathogenicity.In previous study, we isolated a RGS encoding gene designated as mrfA from Monascus ruber, and its deduced amino acid residues shared 87% and 88% identity to the Aspergillus fumigatus FlbA and Aspergillus nidulans FlbA, respectively. In this study, we constructed theΔmrfA mutant; analyzed the morphological development and pigment/citrinin production of this mutant; determined the activities of some key hydrolytic enzymes and antioxidant enzymes of this mutant in fermentation; and studied the effect of mrfA deletion on the mRNA expression level of mgal (G-protein a subunit encoding gene), pksP(Polyketide synthase presumably responsible for pigment synthesis),PksCT(Citrinin polyketide synthase encoding gene), ctnA(Citrinin biosynthesis transcriptional activator), and Chs(Predicted as chitin synthase). The results are as follows: 1 The construction of theΔmrfA mutantPlasmid pCAMBIA3300 was used as a vector to construct the mrfA deletion vector designated as pCMRFA, which was then introduced into Monascus ruber M-7 by Agrobacterium tumefaciens-mediated transformation. In total,153 transformants were obtained and three of them confirmed asΔmrfA mutant by PCR verification and Southern blot analysis were utilized for further studies.2 Analysis of the morphological development and pigment/citrinin production of theΔmrfA mutantThe morphological development and secondary metabolites synthesis of theΔmrfA mutant were compared with those of the wild-type, including hyphal morphology, growth rate, spore production, and pigment/citrinin content. The results showed that the growth rate was significantly reduced in theΔmrfA mutant (p<0.05); when inoculated on PDA medium, theΔmrfA mutant formed a color-reducing colony with tightly compact hypha, which autolyzed from the centre after 7 days of cultivation; no cleistothecium was observed in theΔmrfA mutant, while its conidiospore production was 4.7 times as many as that of the wild-type; when cultivated in PDB medium and incubated at 28℃without agitation, theΔmrfA mutant exhibited delayed germination rate costing 12 hours to complete germination, which was 3 hours longer than the wild-type; when cultured in PDB medium and cultivated at 28℃with continuous shaking at 120 r/min, the wild-type produced 1217.78±120.62 ng/mL citrinin and 4.42±0.16 U/mL (OD390),4.28±0.17 U/mL (OD520) pigments while theΔmrfA mutant produced no citrinin and 0.92±0.04 U/mL (OD390),0.51±0.02 U/mL (OD520) pigments after 10 days of cultivation.Based on these facts, we hypothesized that mrfA played important roles in the regulation of colony growth, sexual reproduction, conidial germination, as well as pigment and citrinin production.3 Determination of the activities of some key hydrolytic enzymes and antioxidant enzymes of theΔmrfA mutant in fermentationPhysiological changes taking place in the submerged cultures of theΔmrfA mutant and the wild-type were studied. Deletion of mrfA caused a significant decrease in the biomass at the early autolytic phase but a dramatical increase in the glucose comsuption rate at the growth phase. TheΔmrfA mutant also showed significantly higher activity of proteinase, chitinase, catalase, and superoxide dismutase than that of the wild-type. The proteinase activity peaked at 7 days of cultivation, and the activity of theΔmrfA mutant was 2.94 U/mL while that of the wild-type was 1.04 U/mL. However, the chitinase activity peaked at 8 days of cultivation, and the activity of both strains was 0.46 U/mL and 0.36 U/mL, respectively. The activity of catalase and superoxide dismutase maintained a constant increase; at 10 days of cultivation, the activity of catalase and superoxide dismutase ofΔmrfA mutant was 13.89 U/mgprot and 483.12 U/mgprot, while that of the wild-type was 5.40 U/mgprot and 268.30 U/mgprot in respective. These results were possibly due to the fact that deletion of mrfA accelerated the autolytic process of M. ruber.4 Role of mrfA played in the transcriptional regulation of genes associated with morphological development and metabolismFull-length cDNA clones of mga1 and mrfA were obtained by RACE technique. The CDS of mga1 is 1062 bp encoding 353 amino acids, while that of mrfA is 2205 bp encoding 734 amino acids.Transcription levels of mga1 and mrfA in the wild-type, theΔmga1 mutant, and theΔmrfA mutant were analyzed by Real-time RT-PCR. The results showed that the expression level of mga1 was upregulated in theΔmrfA mutant at 2 days of cultivation, while it was downregulated at 4,7, and 9 days of cultivation. However, the expression level of mrfA was downregulated in theΔmga1 mutant at 2,4,7, and 9 days of cultivation. In addition, our study suggested that the transcription level of pksP, pksCT, ctnA, and Chs inΔmrfA mutant was lower than that of the wild-type. |
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