Mechanism Of Action Of Dibenzylideneacetone To Inhibit The Chitinase Of Botrytis Cinerea And Its Application Of Cherry Tomatoes Preservation

Botrytis cinerea（B.cinerea）as the main pathogen causing postharvest gray mold of fruits and vegetables,has caused serious economic losses on a worldwide scale.At present,chemical inhibition is still the main way to inhibit the growth of B.cinerea,but with the long-term use of single drug,B.cinerea resistance problems are aggravated,so the need for research and development of new fungal inhibitors is imminent.Chitinase,which is closely related to the synthesis and decomposition of chitin,is an important component of cell wall and cell spacer,and can also produce carbon and energy for fungal growth and reproduction through hydrolysis of chitin,so it plays an important role in the germination of fungi.In this study,we initially screened Dibenzylideneacetone（DBA）,a small molecule with inhibitory effect on B.cinerea,and extracted and purified 40 k Da of B.cinerea chtitinase by prokaryotic expression system,and then verified the inhibitory ability of DBA on purified chitinase activity.By constructing the 3D structure of B.cinerea chitinase,we further explored the mechanism of binding between Chitinase-DBA using computational biology methods such as molecular docking and molecular dynamics simulation.The inhibition and preservation experiments also verified and expanded the application of DBA in real life.Since DBA is a small molecule compound,it can also be used as a precursor substance to synthesize better and more efficient fungal inhibitors,providing new ideas and directions for the opening of targeted gray mold titinase inhibitors.The main research of this thesis are:（1）Screening of chemical inhibitors of B.cinerea and mechanism of action on chitinase.By high throughput screening the small molecule compounds and verifying the candidate compounds to inhibit the activity of chitinase,an B.cinerea inhibitor-DBA with targeted chitinase inhibition effect was screened,and its IC₅₀ value for the inhibition of chitinase activity of DBA was 13.10μg/m L.Subsequently,constructed the3D structure of B.cinerea chitinase was constructed online and several candidate compounds were molecularly docked to the chitinase by Schr?dinger software,and the minimum docking binding energy of DBA was found to be-8.14 kcal/mol.After docking the drug to the active region of the chitinase,molecular dynamics simulations of the composite structure revealed that the RMSD value of DBA was able to remain stable within 200 ns,and the decomposition and calculation of the MM-PBSA binding free energy showed that the chitinase-DBA composite structure was the best bound and the most stable system.By observing the binding pattern within the active region of this complex system,it was found that DBA formed a strongπ-πinteraction with the amino acid residue TYR-243 of the B.cinerea chitinase and a strong hydrophobic interaction with the amino acid residues TRP-382,TRP-135 and ALA-215,which is the reason why DBA can better inhibit the activity of the B.cinerea chitinase.（2）Study on the inhibitory effect of DBA on B.cinerea.The MIC value of DBA was 32μg/m L,and the EC₅₀ values of inhibition of mycelium growth and spore germination were 16.29μg/m L and 14.64μg/m L,and the inhibition activity increased with the increase of DBA concentration.It can be observed by inverted microscopy that the mycelium of B.cinerea treated with DBA will be significantly shortened,crinkled and agglomerated,and the diaphragm will be produced inside the mycelium.Further observation of the mycelium interior by TEM revealed that DBA can cause damage to the cell wall,which has the function of protecting the cell morphology and structure,so DBA can also further cause separation and damage to the cell membrane as well as damage to the organelles,leakage of cytoplasm and hollow phenomenon inside the mycelium.The fluorescence staining experiment further verified that the breakage of cell membrane,leakage of cytoplasm and the accumulation of ROS caused oxidative stress to the mycelium,and all the above test results could verify the actual inhibitory effect of DBA on B.cinerea,thus causing the decay and death of B.cinerea.（3）Study on the effect of DBA on the preservation of cherry tomatoes.DBA was applied to the freshness experiments of cherry tomatoes infected with B.cinerea.It was found that the DBA-treated cherry tomatoes could maintain better sensory qualities such as weight,hardness,color and p H compared with the blank control group,and the correlation analysis also proved the freshness preservation effect of DBA.To sum up,in this study,the chemical-small inhibitor of B.cinerea,DBA,was identified and the mechanism of action between Chitinase-DBA was revealed at the atomic level by a combination of high throughput screening and computational biology.Subsequently,DBA was applied to the model of inhibition and infection of cherry tomatoes by B.cinerea,and the actual inhibition and preservation effects of DBA were investigated,which provided new ideas for the development of new fungal inhibitors and fruit and vegetable preservatives.