| Monascus spp.,produced abundant beneficial secondary metabolites,but some Monascus spp.,produced Citrinin(CIT),a nephrotoxic mycotoxin.Therefore,how to control the CIT in Monascus and its products is particularly important.CIT is a polyketone compound,while pks CT is a CIT polyketone synthase gene.Studies have shown that pks CT gene can produce two m RNA,namely pks CTα and pks CTβ,through alternative splicing(AS)in the transcription process.However,it is not known how Monascus used two m RNAs to regulate citrinin content.In this study,Monascus ruber M7 was used as the research object,and RNA interference and onerexpression techniques were bused to investigate the mutant properties of two mutant that interfered with pks CTα alone and pks CTα and pks CTβ simultaneously.Then the RNAi vector targeting the reverse double promoter structure of mrl3 gene in CIT gene cluster and the overexpression vector of mrl3 gene was constructed.The mrl3 gene interference strains and overexpression strains were obtained by agrobacterium-mediated Monascus transformation,and thecharacteristics of the obtained mutants were analyzed.The main results are as follows:1.Characteristicanalysis ofmutant intron2-2 that interfered with pks CTα alone and exon3-46 that interfered with pks CTα and pks CTβ simultaneousl.pks CTα and pks CTβgenes were interfered by M7,respectively.Colony morphology,microscopic observation,biomass,spore production,pigment and CIT yield of intron2-2 and exon3-46 interfered strains were analyzed.The results showed that pks CT gene interference did not affect the colony morphology,growth and development,spore production and pigment production of Monascus.However,CIT secretion decreased by 94.4% and 79.7%,respectively.Through q RT-PCR analysis,the synthesis of citrinin could be regulated by the expression ratio of pks CTα and pks CTβ.Further transcriptomic analysis showed that the expression levels of serine hydrolase,oxygenase,aldehyde dehydrogenase,oxidoreductase and transporter-related genes levels were decreased.Combined with GO and KEGG pathway analysis,the results showed that Indole alkaloid biosynthesis,Tyrosine metabolism and Phenylalanine metabolism KEGG might be the influencing factors for the difference of citromycin synthesis between the two sample groups.2.Construction of mrl3 gene interference mutant strain and analysis of strain characteristicsThe mrl3 partial gene subjected to RNAi was cloned from M.Ruber M7 genome,and the reverse double promoter RNAi vector p C3300-neo-Ptrp C-EGFP-RNAi-mrl3-Pgpd A was constructed by molecular biological methods such as enzyme digestion and ligation.The interfering strain were obtained by Agrobacterium-mediated erythrobacterium transformation method,and the colony morphology,microscopic observation,biomass,spore producing capacity,pigment and CIT yield of the interfering strains(RNAi42 and RNAi7)were analyzed.The results showed that mrl3 gene interference did not affect the microstructure,growth rate and sporulation ability of Monascus.However,the colony morphology and pigment production ability of Monascus was affected,and CIT secretion ability was decreased by 72.9% and 73.5%,respectively.The expression of mrl3 gene decreased by 87.5% and 88.7%,respectively3.Construction and characterization of mrl3 gene overexpression mutant strain The full-length mrl3 genes were cloned from M.Ruber M7,and the mrl3 genes overexpression vector p C3300-hph-Ptrp C-mrl3-Ttrp C was constructed by molecular biological methods such as enzyme digestion and ligating,,and the overexpression strain was obtained by Agrobacterium-mediated erythrobacterium transformation.The colony morphology,microscopic observation,biomass,spore producing capacity,pigment and CIT yield of the overexpressed strain(mrl3-3)were analyzed.The results showed that the overexpression of mrl3 gene did not affect the microscopic,growth and reproduction of Monascus.The ability of overexpressed strain to synthesize monascus pigment was significantly changed,in which the production of red pigment decreased by 37.8%,while the production of yellow pigment and orange pigment increased by 41.9% and 68.7%,respectively.The CIT production of overexpressed strain increased by 35.8%,36.2% and 26.8% on day 11,13 and 15,respectively.The expression of mrl3 gene in the mutant strain upregulated by68%. |
