| Aspergillus oryzae is a filamentous fungus widely used in traditional brewing industries,mainly in the production of sake and soy sauce.Due to its significant potential to secrete various hydrolases(amylases,proteases,etc.),A.oryzae has been widely used as a host for heterologous expression to improve the production of various hydrolases and secondary metabolites in recent years.In contrast,Aspergillus flavus,a close relative of A.oryzae,has limited application in the fermentation industry due to its ability to secrete aflatoxins.Recently,considerable studies have shown that A.oryzae and A.flavus belong to Aspergillus section Flavi,and their genomes show extremely high similarity in the coding region.A.oryzae originated from a monophyletic branch of a branch of A.flavus in the phylogenetic analysis,and through domestication,A.oryzae was gradually detoxified and differentiated from A.flavus.Nevertheless,no reasonable explanation has been found for the fact that A.oryzae with a complete aflatoxin biosynthesis homologous gene cluster does not secrete aflatoxin.In recent years,the discovery of a large number of aflatoxin synthesis regulators provides a new direction for researchers to explore A.oryzae,which has a complete aflatoxin synthesis homologous gene cluster but does not secrete aflatoxin.Based on this,the evolution of aflatoxin synthesis regulatory genes during the domestication of A.oryzae and whether the regulatory genes can play a functional role in regulating toxin secretion in A.oryzae with a complete aflatoxin biosynthesis homologous gene cluster needs further exploration.The contents of this paper include comparative genomics analysis of A.oryzae and A.flavus,heterologous expression of aflatoxin synthesis regulator genes Stu A and Raf A in A.oryzae and their effects on aflatoxin synthesis,and transcriptome analysis of heterologous expression strains,the results are as follows:1.The genomes of 9 strains of A.oryzae and A.flavus by pan-genome sequencing were compared with the genomes of 3 reference strains for genome feature comparison,gene function annotation,protein prediction analysis,and secondary metabolic gene cluster prediction analysis.The analysis found both conservation and diversity in the number of transporters,carbohydrate-active enzymes,and secondary metabolic gene clusters among genomes of these strains.Phylogenetic tree analysis found that the non-toxin-producing A.flavus and A.oryzae clustered into the same clade,which indicated that the non-toxic A.flavus was more closely related to A.oryzae.Toxigenic A.flavus may have been artificially selected during the domestication process to obtain non-toxic A.flavus,and A.oryzae was probably further evolved from non-toxic A.flavus.For A.oryzae 2351 and A.oryzae RIB40 clustered into the same clade,and A.oryzae 2351 had a complete aflatoxin biosynthesis homologous gene cluster,it was selected as a follow-up study strain to explore its possible strain characteristics.In addition,the sequence alignment of the genes encoding transcription factors Stu A and Raf A in A.oryzae 2351,A.oryzae RIB40,and A.flavus NRRL3357 found that the Stu A and Raf A gene sequences of the three strains had certain differences(base substitution or fragment deletion).2.To explain why A.oryzae with a complete aflatoxin biosynthesis homologous gene cluster does not secrete aflatoxin,the knockout vector p Ao G was transformed into A.oryzae 2351 via using protoplast transformation and obtained the auxotrophic A.oryzae 2351-Δpyr G homozygous strain.Heterologous expression vectors of Raf A and Stu A genes were successfully constructed through ligation and transformation experiments by enzyme digestion and ligation.After further PEG-mediated protoplast transformation,positively heterologous expression strains 2351-Raf A and 2351-Stu A were obtained.3.To explore the morphology of the heterologous expression strains and their response to nitrogen concentrations,each strain was cultured under nitrogen-limited conditions.It was found that the colony of the control strain and the heterologous expression strain did not change significantly due to the change of nitrogen concentration,but the colony of the heterologous expression strain was significantly larger than that of the control strain.The number of conidia of 2351-Δpyr G and heterologous expression strain 2351-Raf A was much lower than that of 2351-Stu A.This result indicated that the transformation of the aflatoxin synthesis regulatory gene Stu A was more conducive to the production of conidia of the strain.In addition,the biomass of the three strains decreased with the decrease of nitrogen concentration,among which the biomass of 2351-Δpyr G decreased most obviously.Compared with the control,mild nitrogen restriction reduced the biomass of 2351-Δpyr G by more than half.The biomass of the heterologous expression strain was significantly reduced under the same nitrogen concentration compared with the control strain.Of them,the biomass of 2351-Stu A was less than that of 2351-Raf A,which indicated that the transformation of Stu A gene had a greater effect on the biomass of the strain.4.To explore whether the heterologously expressed genes Stu A and Raf A can regulate the secretion of aflatoxins from A.oryzae strains with complete homologous clusters of aflatoxin synthesis genes,each strain was cultured under nitrogen-limiting conditions.The toxin assay showed that all the heterologously expressed strains could secrete aflatoxin,and the aflatoxin secretion of the 2351-Stu A was higher than that of the 2351-Raf A.The stronger infection ability of 2351-Stu A in the subsequent experiments of infecting plant seeds confirmed this conclusion again.What’s more,the production of aflatoxin in heterologous expression strains was negatively correlated with nitrogen concentration,indicating that these two regulators Stu A and Raf A may regulate the secretion of aflatoxin via cnotrolling nitrogen metabolism.5.To further explore the molecular mechanism of aflatoxin secretion of heterologous expression strains,the control and heterologous expression strains were subjected to transcriptome sequencing.Different numbers of differentially expressed genes(DEGs)were identified,and differential expression analysis found that the expression levels of the DEGs identified in the aflatoxin synthesis pathway were up-regulated or down-regulated to varying degrees.In total,48 DEGs encoding aflatoxin synthesis-related enzymes were identified in the aflatoxin synthesis pathway.Most of the enzymes encoded by the genes whose expression levels were up-regulated were involved in the synthesis of aflatoxin precursor sterigmatocystin or directly involved in the synthesis of aflatoxin.Additionally,the number of DEGs encoding aflatoxin synthesis-related enzymes in the heterologous expression strain 2351-Stu A was more than that of 2351-Raf A.GO enrichment analysis showed that DEGs were mainly distributed in membrane and membrane part,mainly involved in metabolic and cellular processes,and mainly performed molecular functions such as catalysis and binding.KEGG pathway enrichment analysis showed that the DEGs in the three gene sets were enriched in the pathway of β-alanine metabolism,phenylalanine metabolism,and α-linoleic acid metabolism,respectively.In this paper,aflatoxin synthesis regulatory genes were transformed into A.oryzae with a complete aflatoxin synthesis gene homology cluster.It was found that the regulatory genes can regulate aflatoxin secretion,and the related regulatory mechanism was preliminarily explored through transcriptome sequencing. |
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