Cloning Of The Pigment-producing Gene(s) With Their Functions In Monascus Spp.

Monascus spp., a kind of fungi, used in food production and traditional medicine for a thousand of years in China, has become an important microorganism resources due to its production of many kinds of metabolites, such as monascus pigment, monacolins,γ-aminobutyric acid(GABA), ergosterol and hydrolyase, which are used as food fermentation starter, food colorant, function food and the ingredients or/and raw materials of medicine. At present, the investigation about Monascus spp. was focused on its classification, breeding, metabolites isolation and fermentation conditions to improve useful metablites, but the study on molecular biology of Monascus spp. is at the beginning stage. It was found that most of the sequences related to Monascus spp. in GenBank were used as classification and identification of the homology, few were functional genes. Until now, there was no report about the gene(s) involved in monascus pigment synthesis.In recent years, an efficient method to find and identify new function genes with Agrobacterium tumefaciens-mediated T-DNA transformation has been developed and widely used. In this paper, a transformant library of Monascus ruber by Agrobacterium turnefaciens mediated T-DNA transformation was constructed and more than 5 100 transformants were obtained. From the library, 24 color-producing mutants, which were markedly different from the original strain M-7 in pigment production, were selected, and the colony morphologies, microscopic structures, the stabilities to hygromycin resistance and the abilities to produce different metabolites of the mutants were analyzed. After that, flanked T-DNA sequences of 8 color-producing mutants were isolated by thermal asymmetric interlaced PCR, according to the bioinformation of isolated sequences, a complete cDNA sequence of color-producing mutants named 805~# was coloned with the method of rapid amplification of cDNA end. Finally, the function of the sequence from 805~# was identified by gene knock-out. The main research contents are as follows:1 The construction of transformant library and selection of color-producing mutantsThe transformant library of Monascus spp., containing more than 5 100 transformants was constructed. The results of PCR amplification of transformants by Agrobacterium tumefaciens-mediated T-DNA transformation suggested that all of the genomic DNA of 60 randomly selected transformants were inserted by T-DNA. The results of southern blot revealed that the single-copy rate of T-DNA into the genomic DNA of 24 randomly selected transformants was 70.8％. By morphology observation, 53 phenotype mutants from the library were selected, among which, there were 24 color-producing mutants and their codes were 189~#, 192~#, 533~#, 635~#, 733~#, 805~#, 959~#, 1132~#, 1164~#, 1263~#, 1295~#, 2066~#, 2606~#, 2887~#, 3108~#, 3257~#, 3715~#, 3742~#, 4081~#, 4096~#, 4591~#, 4698~#, 4963~# and 4968~#, respectively. After five rounds of consecutive incubation, except 733~#, 1132~#, 3715~# and 4591~# the rest of color-producing mutants can grow on the PDA containing 20μg/mL of hygromycin and the stable rate to hygromycing resistance amounts to 83.3％.2 Analysis of monascus pigment, monacolin K,γ-amino butyric acid and citrinin of red fermented rice produced by color-producing mutantsThe contents of monascus pigment, monacolin K,γ-amino butyric acid and citrinin of red fermented rice(RFR) produced by 20 stable color-producing mutants, were analyzed by UV-VIS, TLC and HPLC. The results revealed that the mutants rnetabolites described above were different from those produced by the original strain M-7.In the aspect of pigment, RFR of 959~# was of the the highest pigment-value and amounted to 2 712, which was 2.2 times that of M-7, while the pigment-value of 1263~# was the lowest and was 20, which was just 0.01 times that of M-7. In addition, the UV-VIS spectrum and pigment components of 70％of ethanol solution of RFR produced by 20 of color-producing mutants were markedly different from those of M-7. Regarding to monacolin K, the amounts of monacolin K produced by 4081~# was the higest up to 1 601μg/g while that of M-7 was lower than 0.1μg/g. About GABA, the amounts of GABA of RFR produced by 1263~# was up to 6 183.9μg/g and was 14.2 times that of M-7, the amount of 635~# was the lowest to 156.1μg/g and was just 0.33 times that of M-7. With regards to citrinin, the content of citrinin of RFR produced by 635~# was the higest among of all color-producing mutants, which amounts to 154.57μg/g and was 206 times that of M-7, the content of citrinin of RFR produced by 1295~# was the lowest to 0.10μg/g and was 0.1 time that of M-7.According to the pigment-values and yields of citrinin of RFR produced by color-producing mutants, 20 color-produing mutants were classified into four groups: (1)mutants with higher pigment-value and higher-producing citrinin than those of M-7, they were 635~#, 959~#, 1164~#, 2066~#, 2606~#, 2887~#, 4081~#, and 4698~#, respectively; (2)mutants with lower pigment-value and lower-producing citrinin than those of M-7, they were 805~#, 1295~#, 3742~# and 4968~#, respectively; (3)mutants with lower pigment-value while higher-producing citrinin than those of M-7, they were 189~#, 192~#, 1263~# and 4963~#, respectively, (4)mutants with higher pigment-value while lower-producing citrinin than those of M-7, they were 533~#, 3108~#, 3257~# and 4096~#, respectively. 10 mutants, which were 635~#, 1164~#, 2606~#, 805~#, 1295~#, 189~#, 1263~#, 4963~#, 533~# and 3257~#, were selected from above four groups of mutants and used as materials for further investigating the gene(s) related to monascus pigment synthesis.3 Isolation of the DNA sequences flanked T-DNA by TAIL-PCR and analysis of the function of the isolated sequencesThe DNA sequences flanked T-DNA left border of 10 above color-producing mutants were amplified by TAIL-PCR and the DNA sequences of 635~#, 1164~#, 2606~#, 805~#, 3257~#, 189~#, 4963~# and 533~#, were successfully isolated and the length of isolated DNA fragments ranged from 500 bp to 1 300 bp. By FASTA analysis, the amplified DNA from 805~# carries the RGS functional domain of regulator of G-protein signaling, the similarity of which was up to 84 % with that of Aspergillus fumigatus, but the isolated DNA from the rest mutants had no similarity with DNA sequences in GenBank. The isolated DNA sequences from 533~#, 1164~# and 2606~#, which have been published in GenBank, correspondingly, the recorded No. is DQ861336, DQ 861338 and DQ 861337, respectively.4 Identification of the function of DNA sequence coding for regulator of G-protein signaling of Monascus spp.On the basis of bioinformation from isolated DNA sequence of 805~#, the specific primers were designed and the 805~# cDNA sequence with 2 012bp was amplified by the method of rapid amplification of cDNA end. The cDNA sequence carries a long open reading frame with 1 851bp, which includes RGS domain and two DEP(disheveled, Egl-10, pleckstrin) domains. The sequences of both sides of RGS were designed into homologous borders and the mutants with RGS domain deletion was constructed with gene knock-out. The results showed that the mutants with RGS domain deletion produced little hongqu pigment and the isolated flb A positively regulates the hongqu pigment synthesis.