The Study Of Antifungal Activity Of Recombinant Protein Puroindoline A On Grain Molds And Its Stability

Mildew of stored grain is one of the important reasons that cause grain loss and quality decline.Aspergillus glaucs is a typical saprophytic fungus that is active at low water activity and is generally considered a"warning fungus"for early mildew in stored grain.The study found that Puroindoline protein,namely PIN protein,which can effectively inhibit the growth of many plant pathogens,and has obvious resistance to bacteria and mold.In our previous experiments,the Pina gene was cloned from wheat and then expressed the gene heterologously in the prokaryotic cell.In this paper,the recombinant protein PINA was taken as the research object to investigate its antibacterial effect and biochemical stability on the main grain mold,focusing on the antibacterial effect and mode of action of the recombinant protein PINA on A.glaucus.The main results are as follows:The antifungal effects of recombinant protein PINA on five general grain molds,such as Aspergillus candidus,Aspergillus niger,Aspergillus ochraceus,Fusarium graminearum and Alternaria alternata.The results showed that in PDB medium,0.334 mg/mL recombinant protein PINA could completely inhibit the spore germination of A.candidus Link,A.niger,A.ochraceus,and F.graminearum within 12 h,and partially inhibit the spore germination of A.alternata.When using PDA as the medium,0.334 mg/m L recombinant protein PINA can significantly inhibit the hypha growth of the above five grain molds.Among them,the inhibition rates of the rPINA on A.niger,F.graminearum and A.candidus Link reached73.68%,89.13%and 74.5%,respectively.The recombinant protein PINA also showed a significant inhibitory effect on the sporulation ability of A.niger and A.ochraceus.The effects of recombinant protein PINA on germination of Aspergillus aureus spores in CD liquid medium and growth of hyphae on SCDA solid medium were studied.The results showed that 0.09 mg/mL recombinant protein PINA could completely inhibit the germination of A.glaucus spores within 36 h and inhibit the growth of hypha by 25.32%after 5 days of cultivation.When the rPINA concentration was 0.334 mg/mL,the inhibition rate of hypha growth was as high as 80.06%.Then,using A.glaucus as indicator fungus,the antifungal stability of recombinant protein PINA was studied.The results showed that recombinant protein PINA had good thermal stability and could withstand treatment at 70?for 30 min;it maintained the antifungal activity in a buffer solution at pH 6.0-10.0,but was more sensitive to acidic conditions;r PINA was also stable under ultraviolet irradiation,and had antifungal effect in a high-salt environment.For example,it tolerated to K⁺,Mg²⁺,Ca²⁺,Mn²⁺and Zn²⁺at a certain extent and had no significant decrease in antifungal activity treatment with 10%SDS and Tween-20 treatment.Recombinant protein PINA showed a good inhibitory effect on the germination of A.glaucus spores.Scanning electron microscopy observation showed that after treatment with recombinant protein PINA at a concentration of 0.09 mg/mL for 24 h,the spore structure of A.glaucus was deformed,dented,and damaged.By coupling the protein PINA with FITC,the results showed that the FITC-rPINA was present in the spores with a treatment time of 6 h and 12 h.PI staining results showed that the spore cell membrane was damaged and the cell membrane function was lost.The results of JC-1 and DAPI staining showed that the mitochondrial membrane potential was disordered and showed a decreasing trend;the cell nucleus was damaged and the chromosomes were concentrated.The mode of action of recombinant protein PINA on the hypha of A.glaucus was studied.Scanning electron microscopy observation showed that after treatment with recombinant protein PINA at a concentration of 0.09 mg/mL for 24 h,the hyphae showed undesirable changes,such as flattening and wrinkling.FITC-rPINA localization assay demonstrated that after incubation for 6 h and 12 h,the protein PINA was localized in the hypha of A.glaucus.PI,JC-1 and DAPI staining experiments indicated that hypha cells treated with recombinant protein PINA showed loss of cell membrane integrity,decreased mitochondrial membrane potential and DNA damage.This article confirms that the recombinant protein PINA has an inhibitory effect and antifungal stability on main grain molds,and preliminarily explores the inhibitory mode of the recombinant protein PINA on A.glaucus.This research may provide a theoretical basis for the development of rPINA into a grain antifungal agent.