| Nramp(natural resistance-associated macrophage protein)family proteins are a family of transmembrane proteins that are widely distributed in biology and can transport different metal ions,especially divalent trace metal ions,such as zinc,manganese,iron,and copper.As a food-safe fungus,Aspergillus oryzae has been widely used in food fermentation,brewing,and biological agents industries in recent years.Kojic acid,a secondary metabolite of Aspergillus oryzae,is widely used in various fields such as food,medicine,cosmetics,agriculture,and industry becaused of its antioxidant properties,inhibition of tyrosinase activity,antibacterial ability,and anti-cancer effect.The content of this study is consists of the identification of AoNramp1 gene of Aspergillus oryzae,the construction of genetically transformed strains of Aspergillus oryzae,the functional analysis of AoNramp1 overexpression and knockout strains,and the effect of AoNramp1 gene on the synthesis of kojic acid.The research results are as follows:1.To identify the AoNramp1 gene of Aspergillus oryzae,we used bioinformatics methods such as transmembrane domain prediction,multiple sequence alignment,and phylogenetic analysis to identify and analyze the AoNramp1 transporter.Results of the Blast search showed that there is only one Nramp protein family gene in Aspergillus oryzae,named AoNramp1(Ao090003001119).The transmembrane domain prediction and multiple sequence alignment analysis showed that the AoNramp1 transporter contains 11 transmembrane regions.In the multiple sequence alignment,it is found that most Aspergillus fungi have highly similar sequences.There is a consensus transport sequence between the transmembrane regions 8 and 9,which is the consensus transport sequence for the Nramp transporters,indicating that the Nramp protein may have analogous functions in Aspergillus fungi.The phylogenetic analysis showed that the Nramp proteins have high homology among most Aspergillus fungi and are highly conserved.2.The construction of a stable,efficient,and easy-to-operate selection marker for Aspergillus oryzae is the primary consideration for genetic modification of Aspergillus oryzae.In this study,CRISPR/Cas9 gene editing technology was used to knock out the pyr G gene of Aspergillus oryzae 3.042 for constructing an Aspergillus oryzae 3.042 uridine/uracil auxotrophic strain.This work laid the foundation for the further study of the functional characterization of the AoNramp1 gene in Aspergillus oryzae and the exploration of the relationship between the AoNramp1 gene and the synthesis of kojic acid.3.The analysis of the expression pattern of AoNramp1 under different ion treatments showed that the expression of AoNramp1 gene was up-regulated in the control group during the whole process.The expression of AoNramp1 was significantly induced under zinc treatment during 2 to 4 days of cultivation,but inhibited on the 7th day.Similar to the zinc treatment,AoNramp1 was strongly induced and significantly up-regulated relative to the control wild type in the whole manganese treatment.Under iron and copper treatments,there were no significant differences on the expression pattern of AoNramp1 compared with the control during the entire process.To further study the expression pattern of AoNramp1,a 2.0 kb upstream fragment of AoNramp1 start codon as the promoter of AoNramp1 was fused with the GUS(β-glucuronidase)reporter gene to generate a reporter vector.The GUS staining experiment showed that GUS activity was detected in the GUS-PAoNramp1 strain after zinc treatment for 2 days,and the GUS signal became stronger with the extension of the treatment time.After culturing for 4 days,GUS signal was observed in manganese treatment.The GUS-PAoNramp1 strain did not have GUS activity under iron and copper treatments during the entire treatment.The results showed that the expression of AoNramp1 was induced by zinc and manganese.4.To explore the effects of AoNramp1 overexpression and knockout on the growth in Aspergillus oryzae,we used distinct metal ions to treat wild-type,AoNramp1 overexpression and knockout strains.The results showed that the growth diameter and the number of spores in AoNramp1 overexpression and deletion strains were significantly inhibited compared with the wild type strain under CD-Fe treatment.Under zinc,manganese,iron and copper treatments,the growth diameter of both AoNramp1 overexpression and knockout strains were significantly inhibited.In terms of the number of spores,wild-type,AoNramp1 overexpression and knockout strains with copper and manganese treatments shared the same phenotype with the control group.Under iron treatment,there was no significant difference in the number of spores between wild-type,AoNramp1 overexpression and knockout strains.Under zinc treatment,the number of spores of the AoNramp1 overexpressed strain was significantly reduced compared with that of the wild type,while the number of spores of the knockout strain had no significant difference with that of the wild type.Under EGTA and Mn/EGTA treatment,the growth diameter of wild-type,AoNramp1 overexpression and knockout strains was the same,but the inhibition of EGTA to each strain was alleviated by the addition of manganese.In terms of the number of spores,the AoNramp1 overexpressed strain was decreased relative to the wild type,while the knockout strain had no significant difference compared with the wild type.These results showed that the growth of AoNramp1 overexpressed and disruption strains were inhibited5.To explore the effect of AoNramp1 gene on the synthesis of kojic acid in Aspergillus oryzae,we set up three treatments based on the results of GUS staining:control,zinc,and manganese treatments.The results showed that the production of kojic acid in the AoNramp1 overexpression and knockout strains decreased in the control group,but there was no significant difference between them;under the zinc treatment,the output of kojic acid in the wild-type,AoNramp1 overexpression,and knockout strains was significantly improved,and their trend were consistent with that of the control group,and there was no significant difference among the three treatments.In addition,compared with the control group,the yield of kojic acid in the wild type,AoNramp1 knockout,and overexpression strains was increased by 2.6-fold,3.6-fold,and 4.7-fold,respectively.Similar to the control group,the kojic acid production of wild-type,AoNramp1 overexpression and knockout strains are all improved under the manganese treatment.There is a significant difference in the production of kojic acid between the AoNramp1 overexpression and knockout strains,and the wild-type strain.Compared with the control group,the production of kojic acid in wild-type,AoNramp1 knockout and overexpression strains were increased by1.4-fold,1.5-fold,and 1.4-fold,respectively.The results showed that the biosynthesis of kojic acid in Aspergillus oryzae was not affected by the AoNramp1 gene. |
