Study On The Regulation Of Protein Kinase SskB In HOG Pathway On Development And Metabolism Of Aspergillus Flavus

Aspergillus flavus,an opportunistic pathogen,contaminates peanut,maize and other crops with naturally carcinogenic aflatoxins(AFs).There are usually changeable physical and chemical living conditions in the life cycle of eukaryotes.So,many different regulatory mechanisms have been evolved to counteract the diverse environmental stresses.Among them,the pathway responding to osmotic stress named HOG-MAPK pathway has been well studied in some other fungi,but the mechanism in A.flavus is still unclear.Accordingly,in this research,a key protein kinase SskB of HOG pathway was selected to explore its biological function in A.flavus.In this study,the function of SskB protein was mainly studied by constructing the knockout strain(ΔsskB)and the complementary strain(ΔsskB::sskB).The results showed that the loss of sskB gene had no significant effect on vegetable growth.But there was an obvious change in toxin-producing ability,which was nearly completely inhibited.And consisting with the result,the expression of toxicogenic related genes decreased significantly in ΔsskB strain,indicating that the sskB played a key role in the production of toxins.In addition,the number of spores as well as sclerotia and the expression of both regulatory genes decreased remarkably,which fully illustrated sskB playing an important role in the reproductive process of A.flavus.The results of the seed infection test showed that the sporulation of the knockout strain on peanut and maize both decreased,and there was no detectable toxin on the peanut,while a small amount of toxin on the maize,indicating that the gene played a positive role in regulating the pathopoiesia ability of A.flavus.The results of stress experiments showed that the stress resistance of the knockout strain was much weaker than that of the wild type.The inhibition rate of 1 mol/L NaCl was the highest in osmotic stress.Moreover,the response to the cell wall integrity stress was more obvious to the 0.01%SDS,and more evident to the 10 mmol/L H2O2 in the oxidative stress.And the strains were also incubated under different water activity(water activity,Aw).The growth of them was all repressed,especially the colonies,growth under 0.93Aw was almost completely inhibited.All these results indicated that sskB could take part in regulation in many environmental stresses.Meanwhile,the effects of SskB on HogA phosphorylation level of these three strains in different conditions including no stress and 1 mol/L NaCl was detected by western-blotting assay due to its diferently phosphorylated effect.The results showed lower expression level of phosphorylated HogA in knockout strain under normal condition and osmotic stress,suggesting that SskB in stress could indeed make HogA phosphorylated.So,we hypothesized,besides SskB,there might be another protein or pathway not affected by osmotic stress causing the phosphorylation of HogA.To sum up,the results of this study showed that sskB can affect the development,metabolism and pathogenicity of A.flavus,respond to sevaral stress and promote the phosphorylation of HogA.