| Monascus aurantiacus,a traditional Chinese fermentation fungus,is currently the only important microorganism approved for the production of edible pigments in the world.Monascus aurantiacus can produce kinds of useful secondary metabolites,such as Monascus aurantiacus pigments,monacolin K,?-aminobutyric acid(GABA)and ergosterol,etc.The secondary metabolites of Monascus aurantiacus include owering blood fat,lowering blood pressure,antibacterial,anti-cancer aeffects.Monascus aurantiacus also produce citrinin,a mycotoxin harmful to the hepatic and renal systems,which restricts the further application of monascus aurantiacus products.In eukaryotic cells,macromolecular substances are mainly transported through vesicles.As the molecular switches for vesicle traffic.Rab GTPases play important roles in the formation,transportation,and fusion of vesicles.This article intends to study the subcellular localization of the Rab family and the interaction between Rab proteins by constructing p CAmbia-Rab-GFP overexpressing strain to seek the role of the Rab GTPase family in the synthesis of monascus aurantiacus pigment and citrinin,and lay the foundation for the safe and wide application of monascus aurantiacus products.The main contents of this study are as follows:1.Comparative study on autofluorescence of Monascus aurantiacus AS3.4384 and UM28In order to determine the autofluorescence generation time and experimental strains between Monascus aurantiacus AS3.4384 and Monascus aurantiacus UM28,they were cultured in CD medium(CD),CD medium + uridine(CD+U)respectively.The results showed that the autofluorescence generation time of Monascus aurantiacus AS3.4384 was 36-48 h,while the autofluorescence generation time of Monascus aurantiacus UM28 was 24-36 h.Therefore,Monascus aurantiacus AS3.4384 was selected for the following experiment.2.Study on Rab Protein Subcellular LocalizationAfter culturing for 24-34 h,we found that Rab1 was mainly distributed in the tip of the hyphae,Diaphragm of mycelium,and the formed cleistothecium;Rab2 was mainly accumulated in the hyphae and the formed cleistothecium;Rab6A was mainly distributed in the tips and nodes of mycelium;Rab17 was mainly distributed in the tip of the hyphae,the newly grown mycelium,and the newly formed cleistothecium;Rab1,Rab2,Rab6 A,and Rab17 had obvious overlapping signals with the fluorescent dye Synapto Red(?) C2,suggesting that Rab1,Rab2,Rab6 A,and Rab17 were all localized on the Golgi apparatus and membrane organelles.generally involved in membrane transport and other physiological functions.It also further showed that Rab1,Rab2,Rab6 A,Rab17 were generally involved in membrane transport and other physiological functions.3.Rab gene overexpression in Monascus aurantiacus to study on producting citrinin in YES submerged fermentation.Inoculated in YES liquid medium,the overall trend of citrinin content increased first and then decreased.On the 14 th day of YES submerged fermentation,AS3.4384 and Rab1,Rab2,and Rab17 overexpressing strains,the citrinin content reached the highest level,which were 89.52 ?g/m L,58.98 ?g/m L,97.57 ?g/m L,72.71 ?g/m L,respectively.AS3.4384 and Rab1,Rab2,and Rab17 overexpressing strains,on the 14 th day of YES submerged fermentation,the citrinin content reached the highest level,which were 89.52 ?g/m L,58.98 ?g/m L,97.57 ?g/m L,72.71 ?g/m L,respectively.Compared with the wild-type strain,the overexpression of Rab1 and Rab17 reduced the production of citrinin by 34.12% and 18.78%,respectively.After YES submerged fermentation for 16 days,the content of Rab6 A citrinin in Monascus aurantiacus reached the maximum 100.3 ?g/m L.4.Rab gene overexpression in Monascus aurantiacus to study on producting pigments in rice solid-state fermentation.In the solid-state fermentation of rice for 30 days,Inoculated in YES liquid medium,the overall trend of pigment content increased first and then decreased.AS3.4384 and Rab1,Rab2,Rab6 A,Rab17 overexpression strains,the maximum values of monascus aurantiacus yellow pigment were 785 U/g,746 U/g,1148 U/g,887U/g,respectively.Compared with the wild-type strain,the monascus aurantiacus yellow pigment content of Rab6 A and Rab17 overexpression strains increased by 1.46 times and 1.13 times,respectively.In the determination of the orange pigment of Monascus aurantiacus,the maximum pigment content of wild-type strain AS3.4384 and Rab1,Rab2,Rab6 A,and Rab17 overexpression strains were 616 U/g,689 U/g,584 U/g,913 U/g,708 U/g,respectively.Compared with the wild-type strain,the monascus aurantiacus yellow pigment content of the Rab1,Rab6 A,and Rab17 overexpression strains were increased by about 1.12 times,1.48 times,and 1.15 times,respectively.In the determination of monascus aurantiacus red pigment,the maximum pigment content of wild-type strain AS3.4384 and Rab1,Rab2,Rab6 A,and Rab17 overexpression strains were 743 U/g,752 U/g,700 U/g,1023 U/g,752 U/g,respectively.Compared with the wild-type strain,the red pigment content of the Rab6 A overexpression strain increased by about 1.38 times.In the determination of the total monascus aurantiacus pigment content,the maximum values of the total monascus aurantiacus pigment content of AS3.4384,Rab1,Rab2,Rab6 A,and Rab17 were 2132U/g,2291 U/g,2030 U/g,3084 U/g,2420 U/g,respectively.Compared with the wildtype strain,the total pigment content of Rab6 A and Rab17 overexpression strains increased by 1.45 times and 1.14 times,respectively.5.Real-time PCR Study on the Function of Rab Proteins in Monascus aurantiacus AS3.4384The functions of Rab1,Rab2,Rab6 A,and Rab17 proteins were detected by fluorescence quantitative PCR.The results showed that in the Rab1,Rab2,Rab6 A,and Rab17 overexpression strains,the transcription levels of the corresponding Rab1,Rab2,Rab6 A,and Rab17 were significantly higher than those of the wild-type strain,which further showed that the overexpression strain was successfully constructed.On the 16 th day of YES submerged fermentation,overexpression of Rab1,Rab2,Rab6 A,and Rab17 genes can significantly increase the transcription level of citrinin synthesis transcription activator ctn A.However,the transcription level of ctn R decreased significantly,and the relative expression of PKSCT,a gene related to citrinin synthesis,also decreased significantly,indicating that the overexpression of Rab1,Rab2,Rab6 A,and Rab17 would inhibit the total expression level of citrinin after the 16 th day of YES submerged fermentation.On the 12 th and 16 th day of YES submerged fermentation,the overexpression of Rab1 significantly inhibited the transcription level of Rab17,indicating that the interaction relationship may exist between Rab1 and Rab17.On the12 th day of YES submerged fermentation,the overexpression of Rab6 A significantly inhibited the transcription level of Rab17 compared with the wild type,and the overexpression of Rab17 can significantly inhibit the transcription level of Rab6 A.However,on the 16 th day of YES fermentation,the overexpression of Rab6 A can significantly increase the transcription level of Rab17,and the overexpression of Rab17 can significantly increase the transcription level of Rab6 A,therefore,it showed that there may be an interaction between Rab6 A and Rab17. |
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