Functionalanalysis Of Citrinin Flanking Gene CtnR1 In Monascus Sp.M15

Monascus pigments are one kind of natural pigments produced during the growth and metabolism of Monascus spp.It is widely used in the food industry because of its edible safety,good stability,and bright color.However,the citrinin was found as one kind of mycotoxin,which greatly limits the wider application of red yeast rice product.Studies have shown that monascus pigments are closely related to the anabolism of citxin.Exploring the regulated genens network of them,will facilitate the use of gene regulation methods to obtain monascus strains with high yield of pigment but low yield or even no citrinin.Temperature and light could significantly regulate the growth and metabolism of microorganisms.During this thesis,the effects of temperature and blue light on the production of monascus pigments and citrin were systematically studied.It was found that orf7 and ctnR1 genes are closely related to the synthesis of citrinin.And then the function of ctnR1 gene was verified by gene knock-out technique and overexpression.Finally,using high-througput sequencing and digital gene expression profiling,we explored the molecular mechanisms that affect temperature and growth of Monascus spp.The main research contents and results are as follows:(1)M15 was cultured under different temperatures and different intensities of blue light irradiation.It was found that the changes in orf7 and ctnR1 gene expression levels were inconsistent with the other 12 gene changes in the citrinin gene cluster.(2)To verify the role of ctnR1 gene in the synthetic pathway of monascus pigment and citrinin by gene knock-out and overexpression experiments.Deletion of the ctnR1 gene leads to the increased product of secondary metabolites such as pigment or citrinin.The yield of pigments was increased by 3.1 times,and 2.1 times of citrinin.However,the sporulation ability of ctnR1 gene-deficient strains decreased,and the vegetative growth of mycelium became weaker.Overexpression of ctnR1 gene inhibits the product of pigment and citrinin.And after overexpression,aerial hyphae become longer,colony growth rate becomes faster,and spores grow more.It indicated that the ctnR1 gene has a significant negative regulation on the synthesis of pigments and citrinin,and has a positive regulatory effect on the growth and reprocution of the red yeast.Therefore,the ctnR1 gene is an upstream gene for the synthesis of pigments and citrinin.(3)Through high-throughput sequencing,the molecular mechanism of temperature influence on the growth,reproduction,and metabolite production of Monascus spp.The transcript data for Monascus sp.M15 were obtained at 25?,30? and 35?.Differential gene analysis showed that 35? is more suitable for the growth of Monascus sp.and the product of metabolite.At 25?,the product of citrinin increased,while the yield of pigment decreased.In the meanwhile,the metabolism of Monascus decreased and the MAPK signaling pathway was inhibited.The molecular mechanism may be temperature-dependent transcription factors,such as nitrogen metabolism and carbon metabolism,were regulated.The difference in expression was consistent with the KEGG metabolic pathway,indicating that temperature regulates the overall regulatory factor of Monascus sp.M15.