| Monascus spp.are microorganisms for food fermentation.Its fermentation product,red mold rice(RMR)has various physiological activities and a nearly 2000-year history of application in China.Monascus pigments(MPs)are natural pigments produced by Monascus spp.They are mainly used for coloring in food industry.Because of the characteristics of nature and safety,MPs are the food colorants growing fastest in China resently.The study on biosynthesis process of MPs started from early 1960s.But it remains unclear.From middle 1990s,our research team developed molecule biological study of Monascus spp.In 2010,the genom of Monascus ruber(M.ruber)M7 was completely sequenced.A 51 kb sequence of MPs biosynthesis gene cluster was cloned including 16 genes.In this experiment the gene MpigM and MpigN/O from M ruber M7 MPs biosynthesis gene cluster were knocked out by Agrobacterium tumefaciens-mediated transformation(ATMT),respectively.And the positive mutant strains ?MpigM and ?MpigN/O were selected Subsequently,the growth,reproduction and pigments and citrinin production of the mutants were analyzed.The results were as follows:1.Knockout of gene MpigM and MpigN/OHygromycinB resistance gene(hph)was taken as selectable marker to construct gene knockout vectors pCMPIGM and pCMPIGN/O with upstream and downstream sequences of MpigM and MpigN/O respectively.ATMT was taken to transfer M.ruber M7.The obtained transformants were screened and verified by PCR and Southern blot.And the correct mutants?MpigM and ?MpigN/O were isolated.2.Characteristic analysis of ?MpigM and ?MpigN/O2.1 Growth characteristics of ?MpigM and ?MpigN/O on different mediumThe mutant ?MpigM and ?MpigN/O and M.ruber M7 were inoculated on the potato dextrose agar medium(PDA),glycerol(25%)nitrate agar medium(G25N)and czapek yeast extract agar medium(CYA)plates which were usually used to study Monasus spp.and cultured at 28? for 10 d.The colony morphology and microscopic morphology were observed.The results showed that on each medium that the color of ?MpigM colonies was lighter than that of M7.However the colony of ?MpigN/O in various media showed no pigment.In the aspect of growth rate,the colony diameter of ?MpigM on each medium showed no significant difference with M7.On PDA medium,the difference of colony diameter between ?MpigN/O and M7 was not obvious.But on CYA and G25N medium the colony diameter of ?MpigN/O was ouly about 70%of that of M7.The microscopic observation showed that both of M7 and ?MpigM on PDA could produce conidium and cleistothecium.But ?MpigN/O could only be observed the existence of conidium with no cleistothecia on PDA.M7 could produce a large number of conidium on CYA and G25N medium.And the observation results of ?MpigM and ?MpigN/O were coincident with M7.The results above demonstrated that both of MpigM and MpigN/O had signifieant effect on the production of MPs.The deletion of MpigM did not affect the growth and reproduction.However the deletion of MpigN/O would slow down the strain growth rate and affect its sexual reproduction.Thus it was predicted that MpigN/O might be a regulator gene and participate in other reaction besides MPs biosynthesis.Or the multi-cope strain was generated during the gene knockout process,which inactivated other gene.2.2 Comparision of biomassThe spore suspension of M7,?MpigM and ?MpigN/O was inoculated in PDB medium with shaking culture at 28? with 100 r/min.The samples were taken every 2 d.The eulture supermatant and the mycelium were separated by filtration.The mycelium dry weight was measured.According to the mycelium dry weight of different time,the biomass change of the strains was analyzed.The results showed that the tendency of biomass change of ?MpigM and ?MpigN/O was the same with M7.The maximum biomass of M7 could reach 4.7 mg/mL.However the maximum biomass of ?MpigM and ?MpigN/O was 4.3 mg/mL and 3.4 mg/mL respectively.Therefore it proved that MpigM had no influence on the strain growth,and the deletion of MpigN/O would retard growth to a degree.2.3 MPs analysis of ?MpigM and ?MpigN/OThe spore suspension of M7,?MpigM and ?MpigN/O was inoculated in PDB medium with shaking culture at 28? with 100 r/min for 12 d.The MPs in culture supernatant and the mycelium were analyzed by HPLC.The HPLC analysis of ?MpigM showed that the main MPs produced by M7 were not detectable and four kinds of yellow pigments accumulated.Two kinds of them were produced more and analyzed by LC-MS and other method.Combined with relative research they were predicted as Monasfluol A and Monasfluol B respectively.The HPLC analysis of ?MpigN/O showed that no MPs were detected.However in both the culture supernatant and the mycelium another substance was detected which was proved to be initial intermediate of Mps,M7pks-1.According to the analysis of MPs change,it suggested that the protein encoded by MpigM controls the furanone-ring formation dxaring MPs biosynthesis.While the protein encoded by MpigN/O was responsible for the pyran-ring closure.2.4 Citrinin analysis of ?MpigM and ?MpigN/OThe spore suspension of M7,?MpigM and ?MpigN/O was inoculated in PDB medium with shaking culture at 28 C with 100 r/min for 15 d.The samples were taken every 2 d.The culture supermatant and the mycelium were separated by filtration.The citrinin in culture supermatant and the mycelium were analyzed by HPLC.The citrinin production in culture supermatant of M7 and ?MpigM was increased gradually within the 15 d and got the maximum on 15m d,which were 89.3 ?g/mL and 64.2 ?g/mL respectively.While the citrinin production in mycelium of M7 and ?MpigM got the maximum on gth d,which were 6.3?g/mL and 9.5 ?g/mL respectively.The citrinin production in culture supemaatant and the mycelium of ?MpigN/O decreased significantly compared with M7 which was fewer than 0.5 ?g/mL,beyond the detectable rang.According to the results it could speculate that the production process of MPs and citrinin of Monascus spp.were interactional.But the mechanism would be further studied. |
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