Identification Of The Target Protein Of Berberine Against Monilinia Fructicola

M.fructicola can cause brown rot disease in peach trees and other plants,giving rise to considerable agricultural economic loss.At present,management of M.fructicola mainly relies on chemical fungicides which pollute the environment and lead to drug-resistant strains.Biological pesticides are environment-friendly and have ideal antibacterial effects,which have attracted much attention.AS a biological pesticide extracted from C.chinensis,berberine has a significant inhibition on M.fructicola(EC50=8 μg/mL),but the mechanism remains poorly understood.This study explores the underlying mechanism of berberine inhibiting M.fructicola from the following four aspects:(1)In this study,we designed and successfully synthesized a clickable berberine probe which contains an alkyne handle for subsequent click chemistry to achieve conjugation with azide-linked reporter tags so that the target protein was enriched.We tried to fish the protein target of M.fructicola,explore target fishing conditions using this probe and optimize the synthesis conditions of the intermediates rBBR and BBRo.The reaction conditions of microwave synthesis of rBBR were optimized by response surface methodology.When the microwave temperature was 190℃,the microwave time was 7 min,and the solid-liquid ratio was 1:30,the highest rBBR yield occurred.It was found that the best BBRo synthesis relies on NaH as acid binding agent.(2)In order to enhance the binding affinity between the probe and the target protein,we developed a clickable berberine probe which comprises a benzophenone photoaffinity group and an alkyne handle for subsequent click chemistry to mediate the conjugation with azidelinked reporter tags and thereby enrich the target protein.The benzophenone photoaffinity group can form a stable covalent bond with the protein C-H bond under 350 nm irradiation wavelength to augment the affinity of the probe with the target protein.We explored The synthesis methods of intermediates needed for this probe.As a result,BBRi,BBR2,BBR3,A1,A2 and C1 were successfully synthesized.Two synthesis pathways of BBR2 were also explored.BBR2 yield was higher and the cost was lower using 1,2-dibromoethane as the reactant.The efficiency of A2 purification is higher with ethyl acetate/petroleum ether recrystallization.(3)We attempted to fish the target protein of berberine against B.subtilis(G+),K.pneumoniae(G-)and S.cerevisiae(Fungi)using DARTS technology.We investigated the effects of enzymolysis time and enzyme addition amount on the experimental results.The electrophoresis band was strong when enzymolysis time was 15 min,and the concentration ratio of protease to protein was 1:250-1:1250.(4)Enolase(ENO)is a candidate target of berberine against M fructicola.To validate its function,the vector PBI2300-ENO-HPH was successfully transformed into A.tumefaciens.Prior to the transformation of M.fructicola mediated by Agrobacterium,the transformation conditions of co-culture temperature,co-culture time,inducer concentration and A.tumefaciens concentration were systematically optimized.