Study On The Biological Function Of The Protein Kinase SskA In The HOG Pathway Of Aspergillus Flavus

Aspergillus flavus is a common saprophytic filamentous fungus,belonging to the subclass Ascomycetes,Aspergillus.As a pathogenic fungus of human beings,animals and plants,A.flavus induce aspergillosis of human body,which is harmful to the health.And A.flavus also can cause huge economic losses by infecting crops.Aflatoxin,a secondary metabolite of A.flavus,is a carcinogen with strong toxicity and extremely stable physical and chemical properties.Among them,Aflatoxin B1（AFB1）is classified as a primary carcinogen.Therefore,the research on A.flavus and aflatoxin is of great significance to food safety and human health.SskA protein is a homologous protein of Ssk1 regulated by cell hyperosmolar glycerol pathway in yeasts.There is no study on the function of the protein kinase SskA in A.flavus,and its biological function is not clear.In this study,the function of SskA protein in A.flavus was studied by using homologous recombination technology to construct the deletion strain of sskA gene（ΔsskA）and the complement strain（ΔsskA^C）.The results showed that the loss of sskA gene had no significant effect on vegetable growth.However,the number of spores and sclerotia produced by A.flavus decreased and the conidia became sparse.The results of TLC showed that compared with WT,the ability ofΔsskA to produce AFB1 decreased significantly.The results of infection experiment showed that when the sskA gene was lost,the infection ability decreased,especially in the process of infecting peanut and maize.The number of conidia and AFB1 production decreased significantly.In a word,sskA gene played a positive regulation role in the production of conidia,sclerotia formation,AFB1 synthesis and infection of crops.Also,the response ofΔsskA strain to different environmental stresses was explored in this study.The results showed that the strain showed lower resistance under various stress circumstance,suggesting that sskA gene is involved in the regulation and response to various environmental stresses.To further explore the biological function of the Sig＿transdc＿resp＿reg＿receiver domain and two active point D576 and D719 of SskA,ΔsskA^569-734、sskA^D576Aand sskA^K719Astrains were constructed.The results showed that compared with WT,the ability in sporulation,pathogenicity and response to environmental stress of three mutant strains were decreased,but it did not affect the growth and the ability of sclerotium production of A.flavus.The ability of aflatoxin production was affected by the loss of sskA^569-734domain,but D576 and K719 sites in sskA gene did not participate in the regulation of aflatoxin production.In order to prove the effect on A.flavus when two branches of the HOG pathway were both blocked,the double knockout strain（ΔsskAΔste11）was constructed.The results showed that compared with the wild-type strains,ΔsskAΔste11 showed significant down-regulation in conidia production,sclerotia formation,aflatoxin synthesis and pathogenicity.AndΔsskAΔste11 showed lower resistance under various environmental stresses,which suggested that when the HOG pathway is blocked,it seriously affects the normal function of A.flavus.In conclusion,this study preliminarily revealed the main biological functions of SskA protein in A.flavus,and also provides a new idea and theoretical basis for the control of aflatoxin and the reduction of aflatoxin pollution.