Regulatory Mechanism Of CpeB And CatA Genes Of Aspergillus Niger On Oxidative Stress And Fruit Pathogenicity

Aspergillus niger,a filamentous fungus commonly found in nature,has important value in industrial production because it can produce organic acids,enzymes and various secondary metabolites.The fungus can cause the deterioration of food,plants,fruits and vegetables.External oxidative stress leads to large accumulation of reactive oxygen species?ROS?in Aspergillus niger cells.In order to resist oxidative damage caused by ROS,catalase?CAT?and superoxide dismutase?SOD?play important roles in Aspergillus niger.In this study,we used the principle of homologous recombination,combined with protoplast transformation technology,to construct the Aspergillus niger?cpeB,?cpeB-?catA,?cpeB-?sodC deletions and explore the regulatory mechanism of cpeB and catA genes in Aspergillus niger on oxidative stress and pathogenicity on fruit.The main findings are as follows:First,the role of the catalase-peroxidase encoding gene cpeB in oxidative stress and pathogenicity on fruit was investigated.In view of the fact that cpeB gene may be involved in the antioxidant metabolism process of Aspergillus niger,the role of cpeB gene in oxidative stress reaction was investigated by RT-qPCR.It was found that the relative expression level of cpeB gene in Aspergillus niger under exogenous H₂O₂ stress was 3.87times higher than that under normal conditions.Bioinformatics analysis of catalase-peroxidase cpeB revealed that the primary structure of cpeB contains two peroxidase?POD?domains.Meanwhile,by measuring the enzyme activities of CAT and POD of the?cpeB mutant,it was found that the CAT activity of the?cpeB mutant was 22%lower than that of the wild type,and the POD activity was not significantly changed as compared with the wild type.In addition,the?cpeB mutant was subjected to CAT enzyme activity electrophoresis by native-PAGE technique,and it was found that the CAT enzyme activity of the?cpeB mutant was significantly decreased.The above results indicated that cpeB,although structurally a potential POD enzyme,lacks POD activity and has significant CAT activity.At the same time,under the H₂O₂ stress,the mycelium growth of the?cpeB mutant was significantly slower than that of the wild type.The spore germination was determined after incubation for 10 h under 6 mM H₂O₂,and the germination rate of the?cpeB mutant spore was only 17%of wild type.However,the?cpeB mutant has no higher sensitivity to heavy metal ions Cd²⁺,Cu²⁺,Al³⁺and superoxide anion donor menadione,indicating that the Aspergillus niger cpeB gene has a special role in H₂O₂ stress response,while cpeB gene is not required for the cells's adaption to other stresses copper,aluminum and menadione are not required.In addition,during the H₂O₂ treatment,the detection of oxidative metabolism-related substances showed that MDA and ROS metabolites H₂O₂?·O₂^-in?cpeB mutant increased significantly,indicating that the defect of?cpeB mutant in decomposing H₂O₂ leads to membrane peroxidation and ROS damage of Aspergillus niger cells.In view of the fact that Aspergillus niger is a potential pathogen of apple fruit,we investigated whether the cpeB gene in Aspergillus niger was involved in the infectivity of apple fruit.The pathogenicity of the Aspergillus niger wild type and?cpeB mutant was evaluated by inoculating them in apple fruit,and the results showed that the lesion diameter of the?cpeB mutant on the apple fruit was reduced by about 20%compared with the wild type.Further determination of MDA content and ROS metabolites in apple fruit inoculated with Aspergillus niger wild type and?cpeB mutants revealed that apple fruit infecting wild type showed significantly increased content of MDA?H₂O₂ and the rate of production of·O₂^-compared to the?cpeB mutant.In addition,we further determined the expression of CAT family genes in wild type and?cpeB mutant during infection with apples,and found that the CAT encoding genes An02g02750,An03g05660,An08g08920 and cat1,were significantly up-regulated in the?cpeB mutant compared to wild type.These results indicated that the pathogenicity of?cpeB strain on apple fruit was significantly lower than that of wild type.Although the expression levels of other CAT family genes were up-regulated,it still could not compensate for the important function of cpeB,suggesting that cpeB gene played an important role in the pathogenicity of Aspergillus niger on apple fruit.To investigate whether cpeB and CAT encoding gene catA were involved in the oxidative stress response of Aspergillus niger and the virulence of postharvest fruit,as well as the upstream and downstream relationship of cpeB and sodC genes in antioxidant mechanism.?cpeB,?catA and?sodC were used as materials to construct the?cpeB-?catA and?cpeB-?sodC double mutants.By measuring the activity of?cpeB-?catA and electrophoresis of isoenzyme activity,both cpeB and catA had CAT activity,but the contribution of both to the total CAT activity was different.cpeB plays a major role in the total enzyme activity of CAT.In the sensitivity determination experiments of heavy metals,H₂O₂ and menadione,it was found that cpeB and catA genes could participate in the decomposition of H₂O₂,and there was a potential superposition of the function of the two genes.In addition,by analyzing the virulence of?cpeB-?catA,the results showed that simultaneous knockout of cpeB and catA genes did not increase the pathogenicity of Aspergillus niger on postharvest fruit compared to wild-type,?catA and?cpeB.After obtaining?cpeB-?sodC,we also determined the sensitivity of?cpeB-?sodC to heavy metals,H₂O₂ and menadione.It was found that sodC gene was only sensitive to menadione stress,indicating that sodC gene was involved in the detoxification of menadione.In conclusion,CAT and SOD play important roles in the oxidative stress response of Aspergillus niger and the fungal-plant interaction.