Research On Interaction Of NPR1 With TGA2 And Protein Crystallization Of Truncated NPR1

Plants have evolved a variety of defense mechanisms to deal with stress.The invasion of pathogenic can cause the increasing concentration of salicylic acid（SA）in local infected tissues and other systemic tissues,activate and establish a broad-spectrum defense mechanism against secondary infections——systemic acquired resistance（SAR）.Studies have shown that NPR1 can sense the changes in the redox environment caused by the increasing concentration of intracellular SA,monomerizing and incorporating into the nucleus.As a transcription cofactor,NPR1 interacts with TGA to regulate the expression of its own and pathogenesis-related（PR）genes to establish effective SAR.Analysis of the structure of NPR1 can help to elucidate the precise molecular mechanism of SA signaling pathway mediated by NPR1,and provides a theoretical basis for plant disease resistance and breeding research.In this paper,Arabidopsis thaliana NPR1 was used as the research object to study the full length of NPR1,NPR1-TGA complex and truncated protein.The full-length NPR1 protein was obtained by expression and purification in a prokaryotic expression system（E.coli）,which is presumed to be a tetramer and consistent with the existence of intracellular oligomerization;however,it cannot be crystallized due to the uneven state.The NPR1-TGA2/5 complex used to change the NPR1 state was in a highly polymerized state,and the presence of SA did not improve the complex aggregation state,and the complex protein for crystallization could not be obtained.At the same time,the interaction of the C-terminal TGA2 truncated protein with NPR1 also indicates that the interaction of TGA2 and NPR1 is mediated by the C-terminal of TGA2.Although the N-terminal does not directly interact with NPR1,it participates in stabilizing the state.In addition,according to the results of NPR1 digestion and mass spectrometry identification,combining with the prediction of secondary structure,truncated protein crystals were screened and optimized,and obtained△NPR1-42-438 truncated crystals with a diffraction resolution of 2.83（?）.The crystals belong to P2₁ space group with the cell parameter a=56.85（?）,b=147.87（?）,c=238.10（?）,α=β=γ=90.00°.In order to solve the phase problem,Hg co-crystal and selenoprotein crystals were optimized,but the diffraction resolution was not enough to resolve the phase,and optimization was in progress.This article provides a reference for the analysis of NPR1 protein structure and the study of its mechanism of action.