Inhibition Of V-ATPase In The Midgut Of Orientia Armyworms And Prokaryotic Expression Of Wheat Stripe Rust-associated Protein

This study focused on the two proteins in the midgut V-ATPase of the armyworm and purification of wheat stripe rust-associated proteins.It divided into two parts:one is the inhibition of ATP hydrolysis of V-ATPase A and B subunit complexes of the midgut of oriental armyworm;the second is the expression and purification of the wheat stripe rust effect protein(Puccinia Striformis f.sp.Tritici,pst)pst-268,pst-4941,pst-5578 and the target protein Ta ISP.Celangulin V(CV)is a compound isolated from Celastrus angulatus Max that has a toxicactivity against agricultural insect pests.CV can bind to subunits a,H,and B of the vacuolar ATPase(V-ATPase)in the midgut epithelial cells of insects.However,the mechanism of action of CV is still unclear.In this study,the soluble complex of the V-ATPase A subunit mutant TSCA which avoids the feedback inhibition by the hydrolysate ADP and V-ATPase B subunit were obtained and thenpurified using affinity chromatography.The H~+K~+-ATPase activity of the complex and the inhibitory activity of CV on ATP hydrolysis were determined.The results suggest that CV inhibits the ATP hydrolysis,resulting in an insecticidal effect.Additionally,the homology modeling of the AB complex and molecular docking results indicate that CV can competitively bind to the AB complex at the ATP binding site,which inhibits ATP hydrolysis.These findings suggest that the AB subunits complex is one of the potential targets for CV and is important for understanding the mechanism of interaction between CV and V-ATPase.Wheat stripe rust is a devastating disease that seriously affects wheat production and food security in China.Among them,the effector protein in the wheat stripe rust sucker plays an important role in the process of infecting the host.Currently,the pathogenic effector protein cannot retrieve any conserved structure homologous to the effector protein by using the amino acid sequence,so the three-dimensional structure of such effector protein can be analyzed.To provide a basis for further study of the function of this effector protein,an important means of elucidating the pathogenic mechanism of effector proteins from the atomic level.This study investigated three important effector proteins pst-268,pst-4941,pst-5578 and wheat protein Ta ISP.Vector PET-32a-PP was used to express pst-268,pst-4941,pst-5578 and Ta ISP,and PET-32a-PP contained Prescission Protease enzyme cleavage site,which can be expressed.The label of the fusion protein is cut off to facilitate subsequent validation.