Effects Of Nitrogen Sources Metabolism On Growth And Aflatoxins Production In Aspergillus Flavus

Aflatoxins(AFs),produced mainly by Aspergillus flavus and Aspergillus parasiticus,are strongly toxic and carcinogenic.Here,glutamine,as a substitute nitrogen source in Czapek Dox medium,was the greatest AF-producing nitrogen source in A.flavus.Additionally,4 mM glutamine was the threshold for aflatoxin B1 production and growth,but the glutamine synthetase inhibitor had little impact on AF biosynthesis.We also found that rapamycin could suppress the glutamine inducing effect on AFs production,simultaneously inhibit the fungal growth and conidiation.Furthermore,transcriptomes of A.flavus were studied to discover the target genes and regulatory networks of the AFs biosynthesis,especially concerning the nitrogen source metabolism pathway and the target of rapamycin(TOR)signaling pathway.In total,1,429 genes were differentially expressed.Through GO and KEGG pathway analyses,the relationship between nitrogen metabolism and AFs biosynthesis was revealed,and the effects of TOR inhibitor were confirmed.Additionally,the quantitative real-time PCR(qRT-PCR)results verified the credibility and reliability of the RNA sequencing data,and were consistent with the other experimental results.In Aspergillus nidulans,NmrA has been postulated to play a major role in regulating AreA activity.However,the puzzle regarding nmrA function in Asperigillus flavus was unsolved.In the study,we reported the identification and function analysis of the nmrA gene in A.flavus.Our work mainly suggested that the nitrogen metabolite repression regulator NmrA was highly conserved in Asperigillus at the amino acid level,and had structural features of canonical Rossmann fold motif.Moreover,disruption of nmrA displayed repressed growth and aflatoxins biosynthesis mediated by nitrogen sources.We also found that nmrA might play important role in asexual and sexual development because of the increase of conidiation at 29? and sclerotia at 37? in the dark resulting from nmrA defect.Seed infection result indicated that nmrA was required for invasive virulence.In addition,sensitivity tests to various stresses exhibited that AnmrA strains showed the increased sensitivity to rapamycin and methyl methanesulfonate.Furthermore,qRT-PCR analysis demonstrated that nmrA might interact with other nitrogen regulatory and catabolic genes.We also showed that NmrA levels in the cytoplasm and nuclei were modulated in response to different nutrient status in the environment.This research has laid the foundation for primary study on nitrogen regulation network and TOR kinase pathway involving in AFs production of A.flavus.It is also considerable theoretical and practical importances to clarify the regulation mechanisms of AFs biosynthesis and avoid AFs contamination to the greatest extent.