Carbon Catabolite Repression Gene Snf1 And Reg1 Regulate Development And Aflatoxin B₁ Biosynthesis In Aspergillus Flavus

Aflatoxin is highly carcinogenic and toxic and seriously threatens food safety and animal health.Carbon catabolite repressor?CreA?is a pivotal repressor of fungal carbon metabolism and a key node for coordinating the synthesis of hydrolytic enzymes,carbon metabolism and the synthesis of secondary metabolites.Carbon catabolite derepressing protein AMP-activated kinase?SNF1?and protein phosphatase type 1 complex subunit?Reg1?can regulate CreA phosphorylation level and affect the localization and function of CreA.In this study,the regulatory effects of SNF1 and Reg1 on the growth and development of Aspergillus flavus,the secretion of hydrolytic enzymes and the synthesis of aflatoxin were analyzed by constructing the missing mutant strains.The main conclusions are as follows:?1?The colony diameter is generally smaller,the colony color is lighter,and the conidiation lag decreases in the snf1 deleted strain cultured on various carbon sources including glucose,sucrose,trehalose,starch,pectin and microcrystalline cellulose.The colony diameter was smaller,the spore was lower in?Reg1 mutant cultured on glucose,sucrose,trehalose,pectin and microcrystalline cellulose medium.And the conidia formed a complete ring at the edge of the colony in the starch medium.Taken together,SNF1 and Reg1 played an important role in the morphology,fungal growth and development of Aspergillus flavus.?2?Based on the results that the diameter of the?snf1 colony decreases and the diameter of the?Reg1 colony becomes larger on the starch medium,we believe that SNF1 and Reg1 affect amylase expression through CreA.The growth rate of?snf1 and?Reg1 showed a decreasing trend on the culture medium of pectin and microcrystalline cellulose polysaccharide.Therefore,these results suggested that SNF1 and Reg1 might regulate extracellular hydrolase expression by some other pathways.?3?An analysis of the aflatoxin B₁?AFB₁?yield of?snf1 and?Reg1 showed that AFB₁ yield decreased,indicating that SNF1 and Reg1 both had a positive regulatory effect on the AFB₁biosynthesis.?4?The transcriptional levels of aflD,aflM,aflO,aflP genes involved in aflatoxin synthesis and transcription factor gene aflR/aflS and regulation of spore transcription genes brlA,abaA were determined in?snf1 and?Reg1 mutant.All the eight genes showed decreased transcription level when the mutant strain cultured in PDA medium.The results reveal that snf1 and Reg1 affect aflR/aflS,and then the structural genes transcription was inhibited,which eventually caused the toxin synthesis to be blocked.There are CreA binding sites in the aflR/aflS promoter area,SNF1 and Reg1 affect the level of CreA phosphorylation,disrupt the dynamic balance of CreA between nuclei,and affect the transcription expression level of aflR/aflS.In?snf1 and?Reg1 mutants,the genes involved in the regulation of spore formation and development were repressed.In addition,we could find sporogenesis defect based on microscopic observation.Therefore,these results suggest that SNF1 and Reg1 affect the preparation stage of spores,because of the reduction of growth differentiation related gene expression such as brlA,abaA.?5?The effect of SNF1 and Reg1 on the pathogenicity of Aspergillus flavus was studied.It was found that there was little molds growth,and AFB₁ was not detected when?snf1 mutant was inoculated.After?Reg1 mutant was inoculated,less of the surface of peanuts was wrapped by molds,and the content of AFB₁ decreased sharply.We speculate that SNF1 and Reg1 affect the expression of hydrolase,such as amylase and pectinase,and ultimately weaken the aggressive and invasive force.Compare with Reg1,the decrease of infection and localization ability SNF1 deletion mutant was higher.The results of this study lay a theoretical foundation for the risk assessment and prevention and control technology development of aflatoxin pollution.