Functional Preliminary Analyses Of MpigF And MpigP Involved In Pigment Biosynthesis Of Monascus Ruber M7

Monascus spp.are used to produce red fermented rice in China and Southeast Asian countries.Monascus spp.can produce a variety of useful secondary metabolites.Monuscus pigments(MPs)is one of the most important secondary metabolites from Monascus spp..In addition to coloring,some of the MPs also has some biological activities such as antioxidant,anti-inflammatory and anti-cancer.Our laboratory began to engage in the study of molecular biology of Monascus spp.since the mid-1990s.The whole genome sequencing and analysis of Monascus ruber M7 have been accomplished in 2010.In total 16 genes including MpigA to MpigQ have been cloned and the size of this gene cluster is 51 kb.The functional study of genes mainly depends on the genetic engineering methods including deletion,complementation,overexpression and so on.Some MPs gene deletion mutants have been constructed successfully via agrobacterium tumefaciens-mediated transformation(ATMT)method in our laboratory.However,there are still several genes such as MpigP,MpigG,MpigH and MpigI need to be deleted.The purpose of our research is to predict the gene function by the analyses of corresponding gene deletion mutants and complete the pathway of MPs biosynthesis.In this study,we constructed the MpigF and MpigP deletion mutants successfully.Then,The colonial morphology and micromorphology,the production of pigments and citrinin in these two mutant strains and the wild-type strain were studied and analyzed.The results will be described as follows.1.The construction of ?MpigF and ?MpigP deletion mutantsMpigF and MpigP of M.ruber M7 could be expressed at transcriptional level based on the result of RT-PCR.Two casettles of these two genes were constructed via Double-joint PCR method,respectively,and then plasmid pCAMBIA3300 was used to construct gene deletion vector,followed by the A.tumefaciens EHA105 clones(contained gene deletion vector)were induced and incubated for transformation with M.ruber M7 to obtain deletion mutants via agrobacterium tumefaciens-mediated transformation method.Finally,the deletion mutants were verified by the PCR and Southern hybridization.2.The analyses of the growth and development,production of pigments and citrinin of ?MpigF and ?MpigP mutant strainsThe colonial morphology of M.ruber M7,?MpigF and ?MpigP mutants cultured on PDA,G25N,CYA and MA media were analyzed.The results showed that the color of?MpigF was more yellow than M.ruber M7 on PDA and G25N media.The color difference between these three strains can also be observed in CYA and MA media.The color of ?MpigP mutant was significantly lighter than M7 on MA media.The results obtained from micromorphology showed that the conidium and cleistothecium can be regularly generated in these three strains cultured on G25N,CYA,PDA and MA media,respectively.The characteristic of growth and metabolism of the three strains were analyzed after they were cultured in PDB.The results indicated that these two mutants grew slower than M.ruber M7.The pigment constituents produced by the three strains were remarkably different.?MpigF mainly produced four yellow pigments including Monasfluore A,Monasfluore B,Monascine and Ankaflavin.For AMpigP,the pigments mainly included Monasfluol A,Monasfluol B,Monascusazaphilone C and Acetyl-Monasfluol B.The yield of citrinin produced by ?MpigF and AMpigP were reduced sharply.The growth and development of M.ruber M7 were haredly affected in ?MpigF and?MpigP,while the deletion of MpigF and MpigP had significant impact on the production of pigments and citrinin as well as the MPs constituents.Therefore,MpigF and MpigP play important roles in the pathway of Monascus pigments biosynthesis.