Preliminary Study On Function Of Dcl1 And Dcl2 Genes In Aspergillus Flavus

Aspergillus flavus is a saprophytic soil fungus that infects and contaminates pre-harvest and postharvest seed crops.The fungus is also an opportunistic animal and human pathogen causing aspergillosis diseases.Aflatoxins,with strong toxicity and carcinogenicity,are secondary metabolites produced mainly by A.flavus and A.parasiticus.The study of functional genes of A.flavus can help to provide new targets and guidelines for the prevention and control.In this study,DCL1 and DCL2 proteins which are involved in A.flavus RNAi pathway were retrieved by sequence alignment based on the corresponding proteins of Neurospora crassa.?dcl1,?dcl2 and?dcl1?dcl2 mutants were firstly constructed by homologous recombination.Then,growth,sporulation,sclerotium generation,pathogenicity and toxin biosynthesis of the wild type(WT),?dcl1,?dcl2 and ?dcll?dcl2 mutants were analyzed and compared.The comparison of the growth rate with the WT showed that the growth rate of?dcl1?dc12 mutant was consistent with that of the WT,while the growth of ?dcl1 and ?dcl2 strains were inhibited.On the fourth day,the mean diameter of ?dcl1 mutant was 94.17%of that of the WT,and the average diameter of ?dcl2 mutant was 94.63%of that of the WT.There were no significant differences between the three mutants in terms of sporulation,although the sporulation of ?dcll?dcl2 was significantly reduced compared with ?dcll.Contrary,the results showed that sclerotium production of all single-gene mutants were significantly increased comparing with the WT.The production of sclerotium of ?dcl1 and ?dcl2 was increased by 31.91%and 46.13%,respectively.The aflatoxin production was significantly reduced only in ?dcl1?dcl2,in which the average aflatoxin production was only 68.33%of that of the WT.This result suggested that the role of dcll and dcl2 gene on the production oftoxin was redundant.However,the maize-infected assay showed that the conidia number of ?dcl was only 60.44%of that of the WT,and the conidia numbers of ?dcll?dcl2 was only 60.44%of that of the WT.The results of peanut-infected assay were consistent with that of maize.These results indicated that dcl1 was the most important gene during infection.Of significance,the growth of the WT and mutants were studied under different environmental stresses.The results showed that dcl genes had a certain response to osmotic pressure and oxidative stress,but both genes had no significant effect on cell wall stress.For example,compared with the WT,the average inhibitory rate of ?dcll was increased by 7.37%on 1.2 mol/L SOR medium.The average inhibition rate of ?dcl2 was decreased by 11.25%on 8 mmol/LH2O2medium.In summary,in this study,the preliminary understandings of dcll and dcl2 genes on growth,sporulation,sclerotium generation,toxin production,pathogenicity and adaptation to environmental stimuli in A.flavus provided theoretical basis for the storage of grain crops and the prevention from A.flavus.