The Effects Of Histone Deacetylases On The Growth And Secondary Metabolism Of Monascus Ruber M7 Ripening

Monascus spp.are important filamentous fungi.They can produce abundantly beneficial secondary metabolites such as Monascus pigments,Monacolin K and so on These products are used in food and pharmaceutical industries widely.Besides,some Monascus strains also can produce mycotoxin(citrinin),which makes the safety of Monascus-related products doubted.So it is an important issue to use monascus strains to produce Monascus-related products.Histone acetylation can removes the acetyl group from the amino acid residue of core histones to reduce the positive charge,weakening the binding ability of DNA to the nucleosome,which results in the variation of gene expression.Thus,a series of biological response to gene expression occurs.Histone deacetylase is a kind of conserved enzyme and responsible of the deacetylation of core histones?Studies on the function of a variety of histone deacetylases indicated that they play important roles in the growth,reproduction and the regulation of secondary metabolism in many filamentous fungi.According to the annotated DNA information of Monascus ruber M7,there are four genes encoding histone deacetylases.On this basis,We have successfully constructed three gene-deleted mutants(?Mrhda1,?Mrhos2,?Mrsir2),and analyzed the phenotypic characters including morphology development as well as pigment/citrinin production of these strains.The effects of Mrhdal deletion on the mRNA expression levels of nine polyketide synthases also have been evaluated.The results are as follows:1.Construction of ?Mrhda1,?Mrhos2,?Mrsir2 and ?Mrrpd3 strains.According to the results of reverse transcription PCR,four genes of histone deacetylase(Mrhda1,Mrhos2,Mrsir2,Mrrpd3)in Monascus ruber M7 were expressed in the transcriptional levels.The gene-deleted cassettes and gene deletion vectors were constructed,and then the gene deletion vectors were introduced into Monascus ruber M7 by Agrobacterium tumefaciens-mediated transformation.PCR and Southern blot were used to identify mutants.At last,We got the ?Mrhda1,?Mrhos2 and ?Mrsir2 strains.2.The analysis of the growth development and pigment/citrinin production of?Mrhda1,?Mrhos2 and ?Mrsir2 strains.The growth development and the pigment/citrinin production in liquid fermentation of mutants were compared with those of the Monascus ruber M7.With regard to the phenotypic characters,the results showed that ?Mrhda1 strain formed a color-increasing colony on PDA and G25N media.?Mrhos2 strain formed a color-reducing colony on CYA and MA media.On PDA medium,?AdMrhda1 and ?Mrhos2 had no aerial mycelia of peripheral colony.Both of the wild strain and mutant strains could produce conidia on CYA and G25N media,and produce cleistothecia on PDA and MA media.When cultured in PDB medium with continuous shaking,the biomass of ?Mrsir2 strain was higher than the wild strain slightly on each test point.In AMrhdal strain,the production of pigments and citrinin were much higher than the wild strain.On the contrary,the production of pigments and citrinin were much lower than the wild strain in?Mrhos2 strain.The differences of pigments and citrinin production between the wild strain and ?Mrsir2 strain had no obvious tendency.Based on the experiment results,we could draw the conclusion that histone deacetylase played an important role in the regulation of growth,pigment and citrinin production in Monascus ruber M7.Besides,different histone deacetylases played various roles in the growth development as well as pigment/citrinin production.3.Effect of Mrhdal deletion on the transcriptional level of pksSelected mycelium in different culture periods of Monascus ruber M7 and Mrhdal mutant as experimental material.Transcription levels of nine pks genes in wild type strain and ?Mrhdal mutant were analyzed by real-time RT-PCR with the mycelia grown on different period as material.The results showed that the transcriptional levels of genes controlling the biosynthesis of pigments and citrinin were up-regulated in the ?Mrhdal mutant at each measurement time points,while the transcriptional levels of gene GME 1661 were down-regulated,there were no definite trend of transcriptional levels in the rest genes.So we hypothesized that histone deacetylase Hdal has different regulation effects to different pks genes in Monascus ruber M7.