| Monascus spp.are famous for their fermented products,especially red mold rice,a traditional fermented food with a very long edible history documented back to the Han dynasty(BC 202-AD 220).Monascus spp.can produce Monascus pigments(Mps),monacolin K,y-Anminobutyric acid and so on.So Monascus spp.are important producers of natural pigments,antihypertension medicine and anti-hyperlipidemia health food.Mps,as one kind of the main secondary metabolites produced by Monascus spp,are classified as yellow,orange,and red pigments based on their maximum absorption at 380nm,470nm and 505nm,respectively.However,in 1995 France scientisit Blanc,discovered that some Monascus strains could produce nephrotoxic citrinin(CIT),so the safety of Monascus products have become a big challenge.In order to increase Mps content and reduce CIT content in Monascus fermented products,some methods such as screening citrinin-free strains,optimizing culture media and genetic modification have been developed,and some achievements have been made.Light is a very important environment signal which can influence fungal development,primary and secondary metabolites,circadian rhythm and so on.Photoreceptors are protein compounds.Through chromophores,the low-molecular weight compound,photoreceptors can sense different wavelengths of light and let fungi react correspondingly.Up to now in fungi,three kinds of photoreceptors have been uncovered and researched,which are blue,red and green photoreceptors,respectively.Light can also influence many aspects of Monascus spp..The effects of blue and red light on development,MPs and CIT in Monascus spp.have already been reported,but the relative mechanism has less been reported.In this research Monascus ruber M7 is chosen to use as a research object.Firstly,we found one red light receptor gene(fungal phytochrome of Monascus,fphM)and two green light receptor genes(Monascus new eukaryotic opsin,mnop-1,mnop-2)based on genomic sequence information of M.ruber M7,but no any known blue light receptor has been found in M.ruber M7.Then the gene deletion,complement and overexpression strains of fphM,mnop-1 and mnop-2 have been constructed to analyze the effects of red and green lights on development,MPs and CIT contents of theses mutants.The main results were shown as follows.1.Genes and functional domains analyses of red and green photoreceptors in Monascus ruber M 7We found one red light receptor gene fphM and two green light receptor genes mnop-1,mnop-2 in M.ruber M7 using the HMMER program to analyze its genomic sequence,but we haven't found any known blue light receptor such as white collar(wc)genes in M.ruber M7.FphM contains the conserved PAS?GAF?PHY?HisKA?HATPase-C and RRD domains of the typical phytochrome,and one transmembrane domain,but lacks the conserved cysteine residue in PAS domain.Mnop-1 and Mnop-2 contain a conserved bacteriorhodopsin domain and seven transmembrane helixes.Mnop-1 lacks the conserved lysine residue in the seventh transmembrane helixes.Phylogenetic analysis has revealed that Mnop-1 is ORPs-like rhodopsin,Mnop-2 belong to NR-like rhodopsin.2.The effects of different intensities of red and green lights on biomass,Mps and CIT of M.ruber M7The biomass,yellow,orange and red pigments and CIT contents produced by M.ruber M7 were analyzed under 100 lx,300 lx,or 500 lx of red and green light conditions.The results were showed that,compared with M7 under darkness,the biomass was increased about 19%under 300 lx red light,and decreased about 18%under 500 lx green light.The intracellular yellow,orange and red pigments were increased about 7%,9%and 9%;and the extracellular yellow,orange and red pigments were increased about 10%,15%and 15%respectively under 300 lx red light.The intracellular and extracellular yellow,orange and red pigments were dereased under 500 lx red light,300 lx and 500 lx green lights.500 lx green light decreased about 20%intracellular red pigment production in M.ruber M7.100 lx green and red lights had no effect on MPs production.CIT production was decreased under different intensities of red light,with the maximum decrease was 20%.Green light had no effect on CIT production.Since 300 lx red and green lights have the most obvious effect on M7,we chose 300 lx red and green lights as light conditions in the following experiments.3 Construction and characteristic analyses of red and green light receptor genes'deletion,complementation and overexpression strainsHomologous recombination method was used to delete fphM,mnop-1 and mnop-2 in M.ruber M7,and got the deleted mutants:?fphM?Amnop-1 and ?mnop-2,the complementation strains:?fphM::fphM,?mnop-1::mnop-1 Amnop-2::mnop-2 and the overexpression strains:M7::PtrpCfphM?M7::PtrpC-mnop-1,M7:?PtrpC-mnop-2.After 12 days' cultivation at 28 C on the PDA(potato dextrose agar)media,the growth ratioes,microscopic structures,intracellular and extracellular contents of Mps and CIT in above mutants were analyzed and the results were listed as follows.3.1 Characteristic analyses of fphM mutantsThere was no significant difference between all fphM mutants and M7 in growth ratioes and microscopic structures.AfphM had a 16%decrease in biomass,M7::PtrpC-fphM had a 28%increase in biomass.There was no significant difference between ?fphM:fphM and M7 in biomass.For Mps production compared with M7,?fphM had a 9%,15%and 18%increase in intracellular yellow,orange and red pigments production,had a 11%,8%and 17%increase in extracellular yellow,orange and red pigments production,respectively.M7::PtrpC-fphM had a 11%,12%and 18%decrease in intracellular yellow,orange and red pigments production,had a 9%,10%and 9%decrease in extracellular yellow,orange and red pigments production,respectively.There was no significant difference between AfphM:fphM and M7 in MPs production.AfphM had a 50%increase in CIT production.There was no significant difference among M7::Ptrpc-fphM,?fphM::fphM and M7.All these above results indicated that fphM had no effect on development of M7,positive regulate on the biomass,negative regulates on Mps and CIT production.3.2 Characteristic analyses of mnop-1 mutantsThere were no significant difference between all mnop-1 mutants and M7 in growth ratioes,microscopic structures and biomass production.For Mps production compared with M7,Amnop-11 had a 21%,23%and 18%increase in intracellular and had a 17%,11%and 15%increase in extracellular yellow,orange and red pigment production respectively.M7::PtrpC-mnop-1 had a 26%,22%and 26%decrease in intracellular and had a 23%,22%and 31%decrease in extracellular yellow,orange and red pigment production respectively.There were no significant difference between?mnop-1::mnop-1 and M7 in intracellular and extracellular pigment production.?mnop-1 had a 23%increase in CIT production and M7::PtrpC-mnop-1 had a 19%decrease in CIT production.There were no significant difference between Amnop-1::mnop-1 and M7 in CIT production.All these above results indicated that mnop-1 had no effect on development of M7,negative regulate on the Mps and CIT production.3.3 Characteristic analyses of mnop-2 transformantsThere were no significant difference between all mnop-2 transformants and M7 in growth ratioes,microscopic structures and biomass production.For Mps production compared with M7,Amnop-2 had a 29%,20%and 23%decrease in intracellular and had a 19%,14%and 23%decrease in extracellular yellow,orange and red pigment production respectively.M7::PtrpC-mnop-2 had a 23%,26%and 21%increase in intracellular and had a 34%,17%and 16%increase in extracellular yellow,orange and red pigment production respectively.The intracellular and extracellular pigment production between ?mnop-2::mnop-2 and M7 were almost the same.There were no significant difference between mnop-2 transformants and M7 in CIT production.All these above results indicated that mnop-2 had no effect on development,biomass and CIT production,positively regulates the Mps production. |
PDF Full Text Request