Structure And Activity Of Wheat Stripe Rust Effector Protein PST-8853 And V-atpase A B Subunit Complex In Midgut Of Mythimna Separate

Protein is the main undertaker of life activities and participates in various life activities.In this study,we focused on the study of pathogenic effector proteins and proteins in the midgut of V-ATPase of Mythimna separate.This study is divided into two parts:the first part is the expression,purification and structure of the effector protein pst-8853 of Puccinia striiformisone,and the second part is the study of the complex activity of TSCA and B subunits of the midgut V-ATPase and the inhibitory effect of celangulin V on the TSCA and B.Wheat stripe rust has brought serious losses to China’s grain,and the effector proteins in the wheat stripe rust sterilizer have played an important role in infecting the host.However pathogen effector proteins using the amino acid sequence could not find any conserved its homologous.The analysis of the three-dimensional structure of such effector proteins can provide clues for the further study of the function of the effector protein,and explain the important means of the pathogenic mechanism of the effector protein from the atomic level.we studied one of the important effector proteins pst-8853.1.The plasmid pET-32a-pp-pst-8853 containing the gene of interest was obtained by molecular cloning,that inducible expression and found that the effector protein is suspected of zinc binding protein.It was found that increasing the solubility of the protein by adding sarkosyl during protein disruption increases the yield of the protein.Purification methods include Ni-column affinity chromatography and gel filtration chromatography to obtain high-purity proteins,which are then verified by Tricine-SDS-PAGE.2.The characterization of pst-8853 secondary structure by circular dichroism and fluorescence spectroscopy.The structure of the effector protein pst-8853 was found to be altered by the addition of the metal chelating agent EDTA,confirming that the effector protein is a zinc ion binding protein.3.The double-labeled protein samples were analyzed by nuclear magnetic resonance to analyze the three-dimensional structure of the effector protein pst-8853.At present,the main chain spectrum of effector protein pst-8853 was initially assigned using sparky software.The vacuole-type ATPase is an ATP-dependent proton pump,and has important functions such as pH mediation,membrane transport,and invasion of tumor cells in the course of life.The celangulin V can cause insect poisoning reactions.It was found that celangulin V can bind to the Mythimna separate V-ATPase,but the binding site is not yet clear.However,both TSCA and B subunits of V-ATPase are found in the form of inclusion bodies.In this study,the inclusion body was washed and then dissolved with high concentration of urea.Through continuous exploration of renaturation conditions,the soluble TSCA and B subunit complexes are finally obtained and then purified by nickel column.The purified complex was assayed for H~+K~+-ATPase activity,to demonstrate the complex has ATP-hydrolyzing activity.Enzyme activity was measured after adding celangulin V to the complex,and the activity of TSCA and B subunit hydrolyzed ATP decreased.It was confirmed that the TSCA and B subunits in the vacuolar ATPase are one of the targets of celangulin V.This will lay a foundation for understanding the pathogenic mechanism of celangulin V,and at the same time provide assistance for the development of new insecticidal pesticides.