| The filamentous fungus Monacus spp. is mainly used for the production of red fermented rice (RFR). The main secondary metabolites produced by Monacus spp. include Monascus pigment, monacolin K and citrinin and so on. Citrinin is a mycotoxin and nephrotoxic metabolite, which affects the safety of RFR seriously. Therefore, the control of citrinin produced by Monascus is significant. It has been demonstrated that the production of secondary metabolites by filamentous fungi is not only regulated by pathway-specific regulators but also probably by global regulators.LaeA (loss of aflR expression A) is a global regulator in filamentous fungi, which is a methyltransferase and was first found in Aspergillus nidulans in 2004. The homologous gene was also found in other filamentous fungi, such as A. flavus, A. fumigatus and Penicillium-citrinum and so on. The previous studies showed that LaeA regulated vegetative growth, development and production of pigment and mycotoxin in filamentous fungi.The whole genome of Monacus red strain M-7 was sequenced by our lab in 2011, and homologous gene of laeA in M-7 was found through bioinformatics analysis. In order to understand the function of laeA in M. red, we constructed laeA null mutants (ΔlaeA) and laeA over-expression strains (OE::laeA), then analysised the differences in vegetative growth, development and production of secondary metabolites among them. The functional analysis of laeA in Monascus can lay the theoretical foundation of constructing Monascus strains which don’t produce citrinin or produce low content of citrinin.1 Construction of ΔlaeA strains of M. redThe deletion vector pCMRLAEA was constructed, and then was transformed into wild type strain M-7 by Agrobacterium tumefaciens mediated transformation,92 mutants were obtained, and four suspicious mutants were gained by PCR identification. They were respectively numbered 2,6,18 and 22. The No.22 mutant was selected for Southern blot analysis, the results showed that laeA was replaced by single copy hph in the mutant.2 Construction of laeA over-expression strains of M. redThe over-expression vector OEPCMRLAEA was constructed, and then was respectively transformed into wild type strain M-7 and AlaeA strain by Agrobacterium tumefaciens mediated transformation,450 mutants were obtained. No.118 trpC(p)::laeA suspicious mutant and No.106 ΔlaeA::trpC(p)::laeA suspicious mutant were gained by PCR identification. Southern blot results showed that in trpC(p)::laeA strain, single copy trpC(p)::laeA expression cassette was inserted into M-7 strain through non-homologous recombination and there were double copy laeA genes, one was the original, the other one was regulated by trpC promoter. In AlaeA::trpC(p)::laeA strain, single copy trpC(p)::laeA expression cassette was inserted into AlaeA strain through homologous recombination and replaced hph, there was only one copy laeA gene which was regulated by trpC promoter.3 Property analysis of ΔlaeA strains and laeA over-expression strains of M. redThe differences in vegetative growth and development among wild type strain M-7, AlaeA, trpC(p)::laeA and ΔlaeA::trpC(p)::laeA strains were analysed. The results indicated that ΔlaeA strain showed more regular colony edge, longer aerial hypha and faster growth rate than M-7 strain. After incubation on PDA(potato dextrose agar) medium at 28℃ for 10d, the diameter of ΔlaeA strain was 42mm, which was 1.2 times larger than M-7 strain. The number of conidio spore produced by ΔlaeA strain was 5×104/mg dry weight after incubation on PDA medium at 28℃ for lOd, which was as 1.1 times more than M-7 strain. While the number of ascospore produced by ΔlaeA strain was 105/mg dry weight, which was 27% less than M-7 strain. The colony colour and sexual reproduction of ΔlaeA::trpC(p)::laeA strain recovered in some degree compared to ΔlaeA strain, but was not the same as M-7 strain. However, the vegetative growth and development of trpC(p)::laeA strain were the same as M-7 strain. The research results showed that LaeA downregulate vegetative growth and asexual reproduction, but upregulate sexual reproduction in M. ruber.The differences in production of pigment and citrinin during RFR fermentation by wild type strain M-7, ΔlaeA, trpC(p)::laeA and ΔlaeA::trpC(p)::laeA strains were analysed. The results demonstrated that deletion of laeA blocked production of pigment and citrinin, while over-expression of laeA triggered production of pigment and citrinin. After RFR fermentation for 14d, the pigment content of ΔlaeA was 5U/g which was just about 25% of M-7 strain (21U/g). The pigment content of ΔlaeA::trpC(p)::laeA strain was 19U/g that was 10% less than M-7 strain, while the pigment content of trpC(p)::laeA strain was 171U/g, which was 8 times as more as M-7 strain. In the production of citrinin during RFR fermentation, the citrinin content of ΔlaeA strain was undetectable in the whole process of fermentation. The citrinin content of M-7 and ΔlaeA::trpC(p)::laeA strain were accumulated during 6d-14d, while the citrinin content of trpC(p)::laeA strain was accumulated during 6d-12d and reached the top in 12d, then decreased a few in 14d. After fermentation for 14d, the citrinin content of ΔlaeA::trpC(p)::laeA and M-7 strain were respectively 28μg/g and 35μg/g, the production of citrinin by ΔlaeA::trpC(p)::laeA was 20% less than M-7. The citrinin content of trpC(p)::laeA strain was 100μg/g that was 3 times as more as M-7 strain after fermentation for 12d. The research results showed that LaeA upregulate pigment and citrinin production in M. ruber. |
PDF Full Text Request