Study On The Bio-function Of Antioxidant Related Gene Cta1 In Aspergillus Flavus

Aspergillus flavus is a widely distributed pathogenic fungus,which exists in soil,dust and plants.As a saprophytic pathogenic fungus,it has a very strong reproductive capacity and the main harm comes from the secondary metabolite aflatoxins produced by the strain.In particular,aflatoxin B1(AFB1)is the most toxic and carcinogenic natural pollutant in nature,which is extremely harmful to human health and agricultural economy.Therefore,it is of great practical significance to study the growth and development of A.flavus and the regulation mechanism of aflatoxin synthesis.In recent years,many studies have reported that reactive oxygen species(ROS)is an important factor affecting the synthesis and regulation of aflatoxins.Similarly,the pathway of aflatoxins synthesis also undertakes the removal of excess ROS.In order to scavenge ROS,there are primary oxidative defense systems in cells,including catalase,superoxide dismutase,peroxidase,and thioredoxin systems.However,as a secondary antioxidant pathway,the relationship between aflatoxin synthesis pathway and primary antioxidant pathway is still unclear.Therefore,the biological function of catalase CTA1 related to antioxidant activity in A.flavus was studied.In this study,a novel catalase CTA1 from A.flavus was screened by bioinformatics method.The subcellular localization,presence and size of CTA1 were identified by CTA1:e GFP,In-gel enzymolysis mass spectrometry and Western blot.In order to clarify the biological functions of CTA1 in the growth and development,stress response,the level of intracellular ROS,secondary metabolism and pathogenicity of A.flavus,we constructed CTA1 gene knockout strain(?CTA1)and gene complementary strain(?CTA1: CTA1)by homologous recombination method,and determined the related phenotypes.The results showed that the growth and the number of sporulation of CTA1 gene knockout mutate significantly increased on PDA and YES medium,and the length of macro conidiophore significantly increased on PDA.In addition,the absence of cta1 gene significantly enhanced the ability of A.flavus to produce sclerotia and the level of ROS in cells.The results indicated that CTA1 plays a negative role in the growth of A.flavus,the production of conidia,the formation of sclerotia and the level of ROS in cells.Furthermore,the cta1 gene knockout strain was more tolerant under salt ion stress,but more sensitive under oxidative stress.Under the condition of 20 mmol/L hydrogen peroxides and 2 mmol/L t BOOH,the absence of the cta1 gene resulted in a significant inhibiting effect to growth,and the growth of knockout mutant was promoted by 1 mol/L Na Cl and KCl.In addition,under the condition of producing AF,the AFB1 production was significantly decreased(p < 0.01)in ?CTA1 as compared with the WT and ?CTA1:CTA1 strain,and the amount of aflatoxin product of WT strain was significantly decreased under the gradient of high concentration of peroxide.The results of pathogenicity test of A.flavus to corn and peanut seeds showed that the spore yield of ?CTA1 strain significantly increased after infecting seeds,and no aflatoxin AFB1 was produced.Then,the specific mechanism of CTA1 affecting aflatoxin production was further explored.The 138 differentially expressed proteins(DEPs)were found by quantitative proteomics technology,which were distributed in multiple biological processes including biosynthesis process,biological metabolism process,oxidative stress process,etc.KEGG results showed that CTA1 affected aflatoxin synthesis by maintaining ROS homeostasis.These results indicated that CTA1 plays a key role in the growth and development of A.flavus,stress response,intracellular ROS homeostasis,AF biosynthesis and the pathogenicity of A.flavus to crops.To sum up,the function of catalase CTA1 from A.flavus was preliminarily studied in this study.The results showed that CTA1 was involved in the regulation of the pleiotropic phenotype of A.flavus such as hyphal growth,conidia production,sclerotia formation,ROS removal,biosynthesis of aflatoxin B1,response to environmental stress and pathogenicity.