Expression Of Wheatwin1 Protein And Its Inhibitory Effect On Aspergillus Flavus Growth And Toxin Production

Grain Storage Fungi can cause mildew during storage and even produce toxic metabolites.When conditions are right,The fungus especially Aspergillus glaucus,will begin to grow in the grain,the growth and metabolism of Aspergillus glaucus will further create favorable conditions for the growth of other fungi,such as Aspergillus flavus.Aspergillus flavus is the main harmful fungus in the process of grain storage,its growth and metabolism will produce aflatoxin with the ability of carcinogenic and mutagenesis,the four main types of aflatoxins are B1,B2,G1,G2,the most common in food and grain storage is aflatoxin B1.After extracting the whole genome of wheat,the wheatwin1 gene belonging to the fourth family pathogenesis-related proteins(PR4)was amplified by PCR and a mature coding sequence of 378 bp in length was obtained.Double enzyme digestion of wheatwin 1 gene and plasmid pGEX4T-3 using NotI enzyme and EcoRI enzyme,respectively,then they were ligated by T4 DNA ligase and transformed into E.coli competent cells DH5α for cloning.After obtaining the recombinant plasmid T3-W1,it was transformed into E.coli competent cell Origami B for expression and the fusion protein GST-W1 in the form of inclusion body protein was obtained.After the inclusion body protein was refolding,the Glutathione Sepharose 4B gel was used to purify the GST-W1,and then the wheatwin1 was obtained by Thrombin enzyme digestion.Studies on the biological activity of wheatwin1 indicate that it has nuclease activity.Different concentrations of wheatwin1 had certain effects on the spore germination and mycelial growth of Aspergillus flavus,and the effect was significant when the concentration reached 100 μg /mL.Wheatwin1 can promote the increase of aflatoxins at low concentrations.When the concentration reaches 90 μg /mL,it can inhibit the production of toxins by Aspergillus flavus,reduce the accumulation of aflatoxin and have a significant effect.After extracting the RNA of Aspergillus flavus cultured after adding different concentrations of wheatwin1,the five key genes ver-1,aflT,nor-1 aflR,and omtA producing aflatoxin from Aspergillus flavus were subjected to real-time quantitative PCR experiments.Analysis of their expression levels after adding different concentrations of wheatwin1,It was found that the expression levels of the four genes ver-1,aflT,nor-1,and aflR all increased at different levels after adding different concentrations of wheatwin1,the expression level of omtA decreased as the protein concentration increased.Wheat is contaminated with Aspergillus flavus during storage,causing quality damage and loss of reserves,accumulation of aflatoxin in stored grain poses a great food safety hazard,therefore,the prevention of the growth and toxin metabolism of Aspergillus flavus during storage of wheat is of great significance for the safe storage and processing of wheat.This study shows that wheatwin1 has nuclease activity,it has an inhibitory effect on the germination of Aspergillus flavus spores and the growth of mycelium,and it may acts on the cell wall,the accumulation of aflatoxin was significantly reduced at a wheatwin1 concentration of 90 μg /mL,according to the fluorescence quantitative analysis of the five key genes producing aflatoxin from Aspergillus flavus,speculate that wheatwin1 may influence Aspergillus flavus produces aflatoxins by affecting the expression of omtA or other pathways.