Effect Of Water Stress On Growth And Aflatoxin Production Of Aspergillus Flavus

Aspergillus flavus is one of the most important producers of carcinogenic aflatoxins(AF).The development and aflatoxins biosynthesis are regulated by many environmental factors,among which water activity(aw)is one of the most important cues.In this study,the different water activity was mimiced by adding glycerol,and the result showed that the strains exhibited decreased growth,conidiation and aflatoxin biosynthesis under 0.93a_w compared to that under 0.99a_w.When RNA-Seq was used to delineate gene expression profile under different water activities,23,320 non-redundant unigenes,with an average length of 1297 bp,were yielded.By database comparisons,19,838 unigenes matched well with known gene sequences,and another 6767 novel unigenes were obtained by comparison to the current genome annotation of A.flavus.Based on the RPKM equation,5362 differentially expressed unigenes were identified between 0.99a_w and 0.93a_w treatments,including 3156 up-regulated and 2206 down-regulated unigenes,suggesting that A.flavus underwent an extensive transcriptome response during water activity variation.Furthermore,we found that the expression of 16 aflatoxin producing-related genes decreased obviously when water activity decreased,and the expression of 11 development-related genes increased after 0.99 aw treatment.The data corroborated a model that water activity affected aflatoxin biosynthesis through increasing the expression of aflatoxin producing-related genes and regulating development-related genes.In this study,isobaric tagging for relative and absolute quantitation(iTRAQ)technique were used to investigate the effect of aw on proteomic profile of A.flavus.A total of 3566 proteins were identified,of which 837 were differentially expressed in response to aw variations.Among these 837 proteins,403 were over-expressed at 0.99a_w,whereas 434 proteins were over-expressed at 0.93a_w.According to Gene Ontology(GO)analysis,secretion of extracellular hydrolases increased as aw raised,suggesting that extracellular hydrolases may play a critical role in induction of aflatoxin biosynthesis.On the basis of Clustersof Orthologous Groups(COG)and Kyoto Encyclopedia of Genes and Genomes(KEGG)categorizations,we identified an exportin protein,KapK,that might down-regulate aflatoxin biosynthesis by changing the location of NirA.Finally,we considered the role of two osmoticstress-related proteins(Sln 1 and Glol)in the Hogl pathway and investigated the expression patterns of proteins related to aflatoxin biosynthesis.Mitogen-activated protein kinases(MAPK)cascade system of fungi is a long-term evolved regulatory mechanism in response to external environment stimulation.As a member of MAPK,Hog(High osmolarity glycerol)pathway is well known for adapting to osmostress in yeast.In this study,afsakA was knocked out,and we found that growth of afsakA mutant decreased under osmotic stress induced by NaCl and sorbitol.Moreover,mutant showed acute osmotic sensitivity than wildtpye under 0.93a_wand 0.99a_w.When the gene was knocked out,aflatoxin production and conidiation decreased significantly.Hence,the study demonstrated that Hog pathway play important role in A.flavus.It is responsible for adapting environmental water stress.