Validate Functional Of Citrinin Related Genes In Monascus SPP

Monascus as a fermentative strainis was widely used to produce natural pigments and functional food additives in many Asian countries. The pigments have been showed to posses beneficial effects such as anti-inflammatory, anti-oxidative and anti-atherosclerotic properties. In addition, the other secondary metabolites in Monascus also have a variety of regulating functions. However, Monascus has the hazard on the kidney and liver toxic owning to citrinin, which is the bottleneck of the Monascus industry, and then affect the production and export of products. So far, the mechanism of the production and regulation of citrinin has not been clarified. Our research will provide some theoretical basis for the mechanism of citrinin production.In this study, three strains of Monascus were selected because of their phenotype,pigment production and citrinin production. We used seven pairs of primers to amplify the citrinin biosynthetic gene cluster. The sequence of the three strains was identified by DNA sequencing. The similarities were over 99.9 %, which was almost identical with the sequencing released in NCBI. It proved that the gene cluster was the conserved region of Monascus genome.The real-time PCR was used to analyze the expression of related genes in different species of Monascus, and the ctnl, orf6 and ctnRl genes were identified according to the gene information.In this study, we tried to set up gene knock-out approach by Agrobacterium tumefaciens mediated transformation (ATMT) , the orf6 and ctnRl gene were successfully knocked out on the basis of ATMT system. We convert the vector pAG1-H3 into a vector for ctnl, orf6 and ctnRl gene knock out and complement. The general homologous recombination efficiency of ctnRl gene can be as high as 10.5 % and orf6 gene can reach 3 %. The orf6, ctnRl mutants were slightly slowed in vegetative growth rate, the pigment synthesis of orf6 mutant was slightly decreased, the pigment of ctnRl mutant was unaffected, but the ability of producing citrinin improved obviously, mutant strains can be up to 4.5 times higher than the wild; This result predicted that orf6, ctnRl gene as modulator had the feedback inhibition to the citrinin synthesis,we can verify this idea by gene over-expression .