Function Analysis Of Histone Methyltransferase DOT1 In Aspergillus Flavus

Aspergillus flavus,a common saprophytic fungus,produces aflatoxin threatening the human health.Previous studies had been shown that methylation at H3K79 site affected cellular growth,development,secondary metabolism and other related biological activity in all organisms.It is well known that H3K79 methylatio is catalyzed by methyltransferase DOT1 in Yeast.In this study,We identified the histone H3K79 methyl transferase DOT 1 of A.flavus by sequence blast,and proposed that DOT1 putatively participates in the synthesis of aflatoxin.By gene knockout study,the roles of DOT1 in vegetative growth,conidiation,aflatoxin synthesis,and pathogenesis were preliminary illustrated.It was observed that the colony diameter of the dot1 gene knocked out mutant strain ?dot1 is smaller than wild type and complement mutant strain,and the ?dot1 growed more slowly.At the same time,both the number of spores,conidiophores,and sclerotia were increased in the absence of DOT1.In addition,analysis of the production of aflatoxin by TLC and HPLC found that the output of aflatoxin is reduced in ?dot1.Assay of the response to various stress for all strains showed that ?dot1 mutant strain was more resistant to sodium dodecyl sulfate(SDS).Likewise,the ability of ?dot1 mutant resist to antioxidant tertiary butyl peroxide(tBOOH)is increased.And the ability of ?afdotl mutant resist to methanesulfonic acid methylester(MMS),a kind of genome damage agent,was also increased.Assay of infection of plant seeds for both mutant and WT strains showed that the ?afdot1 mutant strain reduced the ability to infect maize seeds in comparison to WT or complementation,which was further supported by counting the number of spore and aflatoxin production.Analysis of the localization of AfDOT1 protein showed that it located in the nucleus.Together,our all data demonstrated that afDOT1 protein palyed important roles,through methylation of histone H3K79 to participate in the regulation of fungal growth,development,biosynthesis and metabolism of aflatoxin,and also played an important role in response to external stress including oxidative stress,cell membrance damage stress and genome damage stress.This study could provide not only the basis data for afDOT1 protein function in fungi,and but also the potential targets for inhibition and reudction of aflatoxin.