Study On Subcellular Localization Of Ctn Synthetic Proteins In Monascus Aurantiacus AS3.4384

The application of Monascus has a long history in China.The use of Monascus as a starter to make wine by the Peiligang people was confirmed about 8,000 years ago.Monascus red pigment,as a secondary metabolite of Monascus,is currently the only natural functional food coloring produced by microbial fermentation,so Monascus is widely used in the world.In recent years,with the development of molecular biology,it has been shown that citrinin produced by Monascus,as a mycotoxin,can seriously damage the kidneys of mammals when ingested too much.It is precisely because of the toxicity of citrinin that severely restricts the development of red yeast rice products.However,the mechanism of Monascus citrinin production has not been fully understood yet.The study provides a theoretical basis for the synthesis and transport of citrinin in Monascus by fluorescence co-localization.In order to deeply explore the localization of the protein encoded by the citrinin synthesis gene in Monascus hyphae,the ctn E,ctn H,orf1 and orf5 genes in the citrinin synthesis gene cluster were selected as the calibration for the study,and the fusion expression vector of citrinin gene and m Cherry was successfully constructed.The citrinin synthesis gene was transferred into the AS3.4384::Rab6A gene fragment in M.aurantiacus by Agrobacterium tumefaciens-mediated transformation.The researches on the dual-fluorescence colocalization system shows that the four citrinin synthetic proteins and rab6A-mediated transporters had the same vesicle structure in the hyphae of M.aurantiacus.It is speculated that the synthesis and transport of citrinin in Monascus is related to the intracellular vesicle transport system.The analysis of citrinin produced by liquid fermentation in shake flasks showed that departure strain M.aurantiacus AS3.4384::Rab6A,a gene that overexpresses citrinin synthesis,leads to increased citrinin production.Compared with the starting strain,citrinin production was increased by 3.2-fold,2.4-fold,2.4-fold and 1.8-fold in the strains containing overexpressed ctn E,ctn H,orf1 and orf5 respectively.Therefore,overexpression of citrinin synthesis gene can promote the production of citrinin by Monascus.Analysis of monascus pigments produced by solid-state fermentation of rice showed that the pigment production of the strains containing Monascus overexpression decreased to some extent.The production of monascus pigment in strains containing AS3.4384::Rab6A::ctn E and AS3.4384::Rab6A::ctn H gene fragments was reduced by 64% and 71% respectively.It can be seen that overexpression of ctn E and ctn H encoding genes has an inhibitory effect on the production of monascus pigment.Statistical analysis shows that there is no significant difference in the production of monascus pigment in the strain containing Rab6A::orf1 or AS3.4384::Rab6A::orf5 compared with the original strain.Therefore,the genes encoding orf1 and orf5 do not affect the production of monascus pigments.Fluorescence quantitative PCR analysis showed that with the prolongation of culture time,the expression levels of the three genes,Monascus pigment-related synthesis genes,sexual reproduction-related genes and asexual reproduction-related genes,will increase under the desicion of Monascus overexpressing citrinin synthesis genes(mpp A,mpp C and mpp E),overexpressing ctn E and orf5 genes and overexpression of orf1 gene in turn.Overexpression of the ctn H gene,which is related to the growth of Monascus,has no significant changes.The expression levels of citrinin synthesis-related genes pks CT and ctn A in both the starting strain and the overexpression strain decreased with the prolongation of culture time.Therefore,the citrinin synthesis gene is not only involved in the synthesis of citrinin in Monascus,but also plays an important role in regulating the growth and development of Monascus and other secondary metabolites.In this paper,the subcellular localization of ctn E,ctn H,orf1 and orf5 genes in citrinin synthesis genes was studied to provide help for further understanding the synthesis and transport of citrinin in Monascus.