Target Confirmation And Mechanism Analysis Of Mulberrin Inhibits Botrytis Cinerea CYP51

Fruit gray mold is one of the devastating plant diseases in the world,which is caused by the infection of Botrytis cinerea.At present,chemical agents play a major part in preventing and combating of Botrytis cinerea.However,the antifungal mechanism of these chemical agents against Botrytis cinerea was still unclear,which limited the research and development of new antifungal inhibitors.Studies have shown that Sterol 14?-demethylase(CYP51)was an important protein that affected the synthesis of ergosterol on the cell membrane of Botrytis cinerea.It could disrupt the cell membrane structure by interfering with the production of CYP51,which led to cell damage.Based on the above research,in this paper,the virtual screening method was used to screen small molecule natural compounds against Botrytis cinerea CYP51,the computer biology and in vitro experiments were used to analyze its mechanism of action and antibacterial effect.In addition,the practical application has been studied and analyzed.Our study provided new research idea and basis for the development of new type of antifungal agent targeting Botrytis cinerea CYP51.(1)Inhibitor screening of Botrytis cinerea CYP51The 3D structure of Botrytis cinerea CYP51 was constructed by I-TASSER 20software and a molecular dynamics simulation of 1000 ns was performed to obtain a stable protein structure.Next,virtual screening was performed in the ZNIC database with CYP51 as the target protein,and the inhibitory activities of 50 natural compounds obtained from the screening were determined.Finally,four compounds:ulberrin,Isobavachin,Isoxanthohumol and Morin were obtained,and their IC₅₀values were 1.480?M,3.872?M,86.070?M,and 150.410?M,respectively.Among them,Mulberrin had better inhibitory effect on Botrytis cinerea than the other three compounds.(2)Research on the mechanism of Mulberrin and Botrytis cinerea CYP51Methods such as molecular docking,molecular dynamics simulation and calculation of binding free energy were used to explore the interaction mechanism of four compounds with CYP51.The results showed that four small molecule compounds could bind to the active area of CYP51.Among them,only Mulberrin could form a?-cation interaction with the Fe ion in the active center of CYP51through the 2-methylpent-2-en group at the C9 atom in its structure,which was a typical competitive inhibition.There was a strong?-?interaction with amino acid residue TYR-113,it also had a closer distance with amino acid residues LEU-116and LEU-498,resulting in a strong hydrophobic interaction.In addition,Mulberrin had the more stronger binding free energy than the other three compounds with CYP51,implying that the 2-methylpent-2-ene moiety at the atom C9 was the critical pharmacophores for the CYP51 inhibitors.Therefore,Mulberrin was selected for the next analysis and research.(3)Study on the antifungal and fresh-keeping effect of Mulberrin against Botrytis cinereaThe minimum inhibitory concentration of Mulberrin against Botrytis cinerea was 32?g/m L.The IC₅₀of Mulberrin toward mycelium growth and spore germination in Botrytis cinerea were 21.049 and 9.480?g/m L,respectively.Inhibition zone experiments showed that when the concentration of Mulberrin was32?g/m L,it was highly sensitive to Botrytis cinerea.By observing the mycelium and its ultrastructure,it was found that after the mycelium being treated with Mulberrin,serious cytoplasm leakage occurred in the cells,and some nuclei and organelles were dissolved and disappeared,indicating that Mulberrin caused certain effects on the cell structure of Botrytis cinerea.Fluorescence staining experiments showed that Mulberrin could destroy the cell membrane of Botrytis cinerea,increased the level of reactive oxygen in the cell,and caused oxidative stress on the mycelium,thereby producing an inhibitory effect on Botrytis cinerea.Finally,the model of Botrytis cinerea infection was used to explore the actual anti-fungal preservation effect of Mulberrin.The results showed that Mulberrin could delay the production of Botrytis cinerea in strawberries,and had a positive effect on strawberry sensory quality,softening and rot rate,weight loss rate,color,hardness and p H.Compared with the BK group and the 1%citric acid aqueous solution group,the 16?g/m L Mulberrin and the 32?g/m L Mulberrin could extend the shelf life of strawberries to the 6th and 9th days,respectively.Therefore,it can be used as the potential natural preserving agent for the storage of the fruit.In summary,Botrytis cinerea CYP51 was used as a research target in this study,combined with computer biology methods,Mulberrin was found to be a new potential small molecule inhibitor of CYP51.The mechanism of Mulberrin and CYP51 was revealed at the atomic level.Meanwhile,in the model of Botrytis cinerea infection,the preservation time of strawberries had been extended through Mulberrin.New research ideas and theoretical basis were provided for the development of new antifungal drugs for fruits and vegetables.