Characterization Of The G-Protein β And γ Subunit Gene In Monascus Ruber

The filamentous fungi Monascus spp., which have been used for the traditional fermented product of red rice in China, can produce a variety of useful metabolites and are widely used in food additives and nutraceutical supplements, but it also cause the food safety problem because of the metabolite citrinin. To further control the content of citrinin, it is necessary to study the regulation of biosynthesis of secondary metabolites in Monascus ruber.The universal signal transduction pathway mediated by heterotrimeric G-protein(Ga, Gβand Gγ) in filamentous fungi plays integral roles for vegetative growth, reproduction and secondary metabolite production. Gβand Gy subunits bound tightly to form a Gβγdimer, and act as a single functional unit during signalling transduction. Previously, the Gβgene (Mgbl) and the Gy gene (Mggl) have been cloned from the fermentation fungus M. ruber in our laboratory. This study through disruption and overexpression of the Mgbl and Mggl to further explore the roles of Gβγin M. ruber, and provide basic theory support on regulation of biosynthesis of secondary metabolites in M. ruber. The major contents were illustrated as following:1. Analysis of Mgbl and Mggl in M. ruberThe gene structure and amino acid sequences were predicted with softberry. Mgbl ORF, including five exons and four introns, was predicted to encode 353 amino acids. The ORF of Mggl, containing three exons and two introns, was predicted to encode 94 amino acids. The predicted Mgb1 protein and Mgg1 protein of M. ruber showed the highest identity with those of Aspergillus fumigatus,96% and 87%, respectively.2. Generation of Mgbl and Mggl deletion mutantsThe Mgbl and Mggl gene replacement vector were constructed using the selective marker hph and neoR, respectively, and then were transformed into Agrobacterium tumefaciens EHA105 via the freeze-thaw method. Three Mgbl deletion mutants(A Mgbl-4,7,23), three Mggl deletion mutants (△Mggl-1,13,17) and three Mgbl/Mggl double deletion mutants (△Mgbl△Mggl-23,34,39) were obtained by the identification of PCR and Southern blot assay.3. Primary studies of Mgbl and Mggl deletion mutantsThe effects of Mgbl and Mggl disruption on vegetative growth, reproduction and pigments and citrinin production were analyzed. Compared with the wild-type strain M-7, all the△Mgbl,△Mggl and△Mgbl△Mggl mutants caused significantly reduced vegetative growth on potato dextrose agar (PDA) plates, and failed to develop cleistothecium on PDA and MA. These results suggested that the G(3 and Gy are necessary for vegetative growth and reproduction of M. ruber.For 16 days cultivation in YES liquid medium, the wild-type strain M-7 produced 8.82±0.41 U/mL pigments (OD485 nm), whereas△Mgbl,△Mggl and△Mgbl△Mggl mutants produced 19.41±0.90,20.46±0.29 and 38.83±0.52 U/mL pigments (OD485nm) respectively; and M-7 produced 21.46±0.25μg/mL citrinin, whereas△Mgbl,△Mggl and△Mgbl△Mggl mutants produced 38.80±2.56,41.27±2.76,34.70±1.23μg/mL citrinin respectively. These results suggested that both Gβand Gy were involved in the production of pigments and citrinin.4. Generation of Mgbl and Mggl overexpression vectors and primary studies of mutantsThe Mgbl and Mggl overexpression vectors pCNB and pCHG were constructed using the selective marker neoR and hph, and transformed into M-7 and each deletion mutant by A. tumefaciems mediated transformation, respectively. The colony growth, cleistothecia formation and conidia formation of NB-M7, NB-△B, HG-M7 and HG-△G mutants were compared with M-7, and no significant differences were found. After 16 days of YES cultivation, the yield of citrinin in NB-M7, NB-△B, HG-M7 and HG-△G mutants were approximately 50% of that in M-7. But statistical analysis showed that the mutants have equivalent ability to produce pigment with M-7.