Functional Analysis Of Carbon Catabolite Repression Gene CreA In Aspergillus Flavus

Aspergillus flavus is a kind of saprophytic fungi,which is pathogenic to important agricultural crops,such as living peanuts and maize.Harm caused by A.flavus lies mainly in its secondary metabolites-aflatoxins.Aflatoxin is a highly toxic substance,commonly seen in nature,which can damage human and animal liver cells,cause liver cancer and even death.Carbon catabolite repression is a very important mechanism in the utilization of carbon as an energy source,required for the regulation of growth,development and secondary metabolite production in fungi.This mechanism has been widely studied in filamentous fungi,most especially,the model fungus Aspergillus nidulans and was found to act through creA,a negative transcriptional factor that inhibits the utilization of secondary carbon sources.However,little is known about the major carbon catabolite repressor gene creA in Aspergillus flavus.Hence,we employed the method of homologous recombination to construct creA gene deletion and over-expression mutants of Aspergillus flavus to explicate the role of creA in the morphology,pathogenicity,expression of genes related to the production of secondary metabolites and secondary metabolite production in A.flavus.We investigated these factors using three different carbon sources:glucose,sucrose,and maltose.Our results showed that not only the gene,but also the quality of carbon source is important in the morphology,pathogenicity and secondary metabolite production of A.flavus.In the growth experiment,we used two different media;PDA,MM+2%D-glucose,MM+2%sucrose,MM+2%maltose,to culture the wild-type,creA gene deletion and over-expression mutants(WT,?creA,and OE::creA,respectively).We observed that the growth of ?creA on glucose was better than on sucrose.However,the growth of ?creA on maltose was better than on glucose,but worse than on PDA.In the observation of conidia production,we found that ?creA produced less conidia and fewer conidiophores than WT.While OE::creA produced more conidia and conidiophores than WT.The expression levels of the genes abaA and brlA,involved in conidia production were lower in ?creA than WT;but higher in OE::creA.In the cell-surface hydrophobicity analysis,we observed that water was absorbed by ?creA.Based on the relationship between cell-surface hydrophobicity and hyphal formation,we suggest that creA is involved in hyphal formation.When grown on PDA medium,we extracted less or no aflatoxin in ?creA.In the analysis of sclerotia production,we observed that ?creA produced more sclerotia than WT;and sclerotia were at the center of the colony,while OE::creA produced less sclerotia than WT.The qRT-PCR results of sclerotia producing genes nsdC,nsdD and sclR revealed:the expression levels of the three genes were higher in ?ceA than WT,but lower in OE::creA.In the stress experiment,we found that ?creA was more sensitive to salt and oxidative stress than WT when grown on PDA medium containing salt and hydrogen peroxide;it was also found on PDA at different temperature.?creA was more sensitive to cell wall stress than WT when grown on PDA medium containing SDS and calcofluor white.However,OE::creA was more sensitive to salt and oxidative stress than WT when grown on PDA medium containing salt and hydrogen peroxide,this was also found on PDA at different temperature.OE::creA was a little sensitive to cell wall stress than WT when grown on PDA medium containing SDS and calcofluor white.In the analysis of seeds infection,we found that ?creA was less pathogenic and produced less aflatoxin than WT.In the subcellular localization experiment of CreA protein,we discovered that CreA was located all through the cell,and also that carbon sources affected the subcellular localization of CreA protein.In the study,we concluded that the gene creA was related to growth,conidia production,aflatoxin production,sclerotia production and pathogenicity in A.flavus.Deleting creA,the mutant produced less or no aflatoxin,less conidia and more sclerotia than WT.Most importantly,its pathogenicity to seeds was reduced.In this study,we analysed the functions of creA through the growth and pathogenicity experiments on mutant strains,laying a theoretical foundation for the biological control of Aspergillus flavus,and providing solutions to the pathogenicity of Aspergillus flavus.