| Aflatoxin is a serious threat to human and animal health due to its immunosuppressive,teratogenic,carcinogenic and mutagenic effects,which are mainly produced by Aspergillus flavus.Therefore,preventing the germination of Aspergillus flavus spores is an effective way to inhibit aflatoxin contamination.So,the research has vital scientific significance to inhibit spore germination and growth and further prevent aflatoxin contamination by identifying the process of metabolic pathways and regulatory pathways in Aspergillus flavus spore germination.In this study,the morphological changes of Aspergillus flavus spores were observed at the different stages of germination,biological technologies such as RNA-sequencing and metabolomics were used to learn the key metabolic pathways and genes during spore germination,and the feasibility and mechanism of controlling Aspergillus flavus spore germination were explored by exogenous substances.The result can provide a scientific basis for the prevention aflatoxin contamination.The main work is as follows:1.Morphological changes during spore germination of Aspergillus flavusIn this experiment,the germination rate of spores was studied by inoculating Aspergillus flavus spores in Czapek culture medium after 0,2,4,6,8,10,12 h,and the morphological changes during the germination process were observed by different microscopes.The results are as follows:1)The germination rate of Aspergillus flavus spores increased exponentially with the culture time.Spores began to germinate after 6 hours of culture in medium,with an average germination rate of 1.86%;from 8 h to 12 h,the germination rates were 16.19%,49.16% and 85.83%,and increased significantly.The results showed that the germination of Aspergillus flavus spores is a gradual process,which is controlled by the culture time.2)The different stages of germination have different morphological characteristics.In the dormancy stage,the spores have a multi-layered folded cell wall structure,which is resistant to the external extreme environment.After spore germination,the cell wall became smooth,the intracellular vacuole gradually expanded and divided.The water acted as a continuous phase to promote the dissolution and transport of various nutrients,providing an environment for nutrient metabolism after spore germination.At the late stage,the nucleus began to divide and the cells entered the division stage,gradually forming mycelia to complete germination.2.The key metabolic pathways during spore germination of Aspergillus flavusIn this experiment,Aspergillus flavus spores were cultured for 0 h,4 h,8 h,and 12 h in CZ medium,and the key metabolic pathways of spores at different germination stages were studied by transcriptomics and metabolomics.The results are as follows:1)The transcriptomic results showed that the differential genes at different germination stages had significant characteristics.In the early stage of germination,the spores entered an active state from a dormant state,with enhanced respiration,intensified energy metabolism,and began to synthesize amino acids,lipids and other substances.This stage had the largest number of differential genes;in the middle stage of germination,spores began to divide,and the main differential genes are nutrient transport-related genes;in the late stage,the differential genes were also mainly nutrient transport-related genes,and the expression of secondary metabolism-related genes continued to be up-regulated.2)The metabolomics results showed that the metabolites at different germination stages had significant characteristics.In the early stage,the metabolites with the largest differential fold change were fructose,glucose,and pine triose,the main difference of different sugars;in the middle stage,the metabolites with the change were 3,4-dihydroxyphenylacetic acid,sulfur Amines and 2-hydroxy-4-methylvaleric acid were mainly the changes of organic acids and phenolic acids;in the late stage,the metabolites with change were glyceraldehyde phosphate,Lys phosphatidylcholine,2-hydroxyethyl phosphonic acids,mainly organic acids and lipid changes.3)The combined analysis of transcriptomics and metabolomics showed that in the early stage of spore germination,the main enriched pathways of differential metabolites and differential genes were amino sugar and nucleotide metabolism,starch and sucrose metabolism,focusing on amino acid synthesis and energy metabolism;in the middle stage,the pathways are pentose and glucuronic acid metabolism,mainly the difference in energy metabolism;in the later stage,the pathways are carbon metabolism,glycolysis process,pentose phosphate metabolism and pyruvate metabolism,the main difference in energy metabolism.3.Carbon and nitrogen metabolism during germination of Aspergillus flavus sporesBased on transcriptomics and metabolomics,this experiment studies the carbon metabolism-related genes and nitrogen metabolism-related genes of spores during germination,to study their effects on the germination rate and related metabolic genes of different wild-type and mutant strains.The result is as follows:1)During the germination of spores,it is selective for different carbon sources.Compared with fructose and glucose,sucrose and trehalose significantly promoted the germination rates by increasing the expression of polysaccharide synthase genes.but glucose can significantly increase the cell size of spore germination during germination.2)The nitrogen metabolism-related genes of s spores changed significantly during the germination process.MepA、MeaA、CrnA、NiiA and NiaD genes all increased significantly at the beginning of germination but did not change significantly later.The gene MepA and MeaA were knocked out in Aspergillus flavus,and the mutant ΔMepA was obtained,the ΔMeaA mutant can not survive.Compared with the wild type,the germination rate of ΔMepA was significantly lower than the wild type in different nitrogen sources,and nitrate was more suitable for the spore germination.The expression of MeaA gene in ΔMepA was significantly up-regulated.The results showed that MepA and MeaA jointly played the role of ammonium transporter,among which MeaA was more critical.4.Effect and mechanism of exogenous substances on spore germinationIn this experiment,essential oil(coumaric acid and geraniol)were selected as exogenous additives to study the effect and the regulation of key pathways during germination.The results are as follows: both coumaric acid and geraniol have certain inhibitory effects on the spore germination,and 200 mg/L coumaric acid and 100 mg/L geraniol were better;Essential oil significantly reduced the cell size of spores when cultured for 8 h;the activities of ROS and antioxidant enzymes were significantly increased in the essential oil treatment group,and the activities of oxidoreductases were also significantly increased;among nitrogen metabolism-related genes,MepA and MeaA gene expression were significantly down-regulated,while NiiA and NiaD gene expression was significantly up-regulated.Overall,the conclusions are as follows: 1)The germination rate is time-dependent,and different germination stages have different morphological characteristics.During the germination process,the spores first absorb water and expand to form a continuous phase of water to initiate the germination process,gradually undergo cell division to complete the germination.2)Spores have different metabolic characteristics in different stages of germination,and the metabolic changes are the most important in the early stage of germination.Protein synthesis and respiration play an important role in the initial stage of spore germination.In the middle and late stages of spore germination,the ways of energy metabolism begin to diversify,accompanied by the synthesis of secondary metabolites.4)Spores are selective for different carbon sources during the germination process.Compared with fructose and glucose,sucrose and trehalose are more suitable for the germination of spores by promoting the expression of polysaccharide synthesis genes;The nitrogen source is also selective,and nitrate is more suitable for spore germination than nitrite and ammonium.In the nitrogen metabolism pathway,ammonium transporter genes(MeaA and MepA)play an important role in the germination process,among which MeaA is the most important ammonium transporter.4)Coumaric acid and geraniol can significantly inhibit spore germination by affecting nitrogen metabolism-related genes and redox balance during spore germination of Aspergillus flavus. |
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