Establishment Of PDHc E1 Expression System And Enzyme Activity Assay Of Fusarium Graminearum

As an important food crop,wheat accounts for one quarter of the total cultivated area of grain in China and the total production of wheat is less than that of rice.Fusarium head blight(FHB)is one of the most important diseases in wheat production,which leads to a significant decline in wheat quality and yield.In recent years,FHB in China is more and more serious with a tendency from the south area to the north area,which has brought great threat to the safety of wheat planting and agricultural development of our country.The pathogen causing FHB is Fusarium graminearum.In China,the chemical control is the main method for the prevention and control of FHB.Because long-term irrational use of drugs,the frequency of resistance to carbendazim in FHB pathogens is seriously.Therefore,it has great significance to develop a new fungicide with a new target and no cross-resistance with existing fungicides in agricultural production.The pyruvate dehydrogenase complex is responsible for regulating energy metabolism in the body.Pyruvate dehydrogenase,a key rate-limiting enzyme in the pyruvate dehydrogenase complex,plays an important role in the entire physiological metabolic pathway.PDHc E1 has high specificity among different species,which can be used as a potential target of new pesticides.Therefore,it is expected to obtain new fungicide candidate compounds by screening PDHc E1 inhibitors.In this paper,prokaryotic and eukaryotic expression systems were established with the target of PDHc El protein from Fusarium graminearum,and the bioactive protein was successfully obtained.Then,we successfully screened bioactive compounds with inhibitory activity against PDHc E1 which synthesized in the early stage of our laboratory.The specific research contents include:Firstly,two recombinant plasmids,pET32a-PDHcE1α and pET32a-PDHcE1β were constructed by single-gene homologous recombination,and the prokaryotic expression system was established.The α and β subunits of the enzyme were obtained in vitro by AKTA purification system.The expression of the enzyme was best when the induction temperature was 20℃,the expression time was 16 h,and the IPTG concentration was 0.1 mM/L.The non-specifically bound heteroprotein was further eluted with 20 mM imidazole,and the specificity was eluted with a high concentration of 250 mM imidazole.The bound target protein is purified to obtain a higher purity target protein,but the protein has no biological activity.Secondly,the eukaryotic expression system expressing PDHcEla and PDHcE1βprotein in Gibberella was constructed by using Gibberellin transformation method,and the target protein was successfully purified by S-tag in protein.Western blotting test proved that the target protein we obtained was presented by the heterotetramer.The specific activity of the enzyme was determined by spectrophotometry to be 4.23 U/mg.Finally,base on the preliminary work of the laboratory,the compounds with high inhibition of pyruvate dehydrogenase were screened out.Compound 1 showed the best inhibitory effect against pyruvate dehydrogenase with an IC50 value of 0.45 ± 0.13 μM/mL.The molecular interaction between compound 1 and pyruvate dehydrogenase protein was determined by isothermal titration calorimetry to elucidate the mode of interaction between the active compound and protein of PDHc El.Therefore,this work provides a scientific basis for the subsequent structural design and optimization of new PDHc E1 inhibitors with high selectivity and high activity.