Salicylic Acid Receptor NPR1 In Tomato:Expression,Purification And Crystallization

Pathogens,microbes,etc.have a tremendous impact on the growth of plants.Plants have the ability to become immune during long-term evolution,forming an immune system capable of targeting exogenous invaders.Usually divided into two categories,one is on the outer surface of cells,triggered by pattern-recognition receptors induced by microbial-associated molecular patterns and damage-associated molecular patterns;the other is in cells,caused by toxic effectors released by pathogens The effector triggers immunity.The important hormone in both immune systems is salicylic acid.Salicylic acid is predominantly perceived through its receptors NPR1,NPR3/NPR4,which in turn functions,and NPR1 is a high-affinity salicylate-binding protein whose binding activity is required for salicylic acid-induced immune function.The evolution and maintenance of different salicylate receptors is likely due to the need for complex controls over the salicylate response.When salicylic acid levels are low,NPR3/NPR4 inhibits defense gene expression,which prevents autoimmunity.Increased accumulation of salicylic acid eliminates repression and allows further induction of defense gene expression by the transcription coactivator NPR1.However,how NPR1 regulates the transcription factor and transcription factor binding to regulate the expression of downstream genes,and the mechanism of binding to salicylic acid is not currently clear.This paper hopes to explain the regulation mechanism of NPR1 by analyzing its structure,and hopes to further elaborate the specific mechanism of the perception and signal regulation of salicylic acid.In this thesis,the tomato NPR1 protein was studied.When the protein was expressed,two expression modes were used: prokaryotic expression and eukaryotic expression.In prokaryotic expression,a protein expression vector for NPR1::pET-28 a was constructed,and proteins were induced at low temperature and low speed.Using this expression format,no crystals were obtained after protein purification.The following optimized conditions were used: increasing the pH of the buffer and increasing the ability to solubilize the protein;increasing the imidazole concentration of the eluted protein to 50 mM and 70 mM,respectively;using ion exchange to ensure the nature,distribution and strength of the charge carried by the protein;Gel filtration chromatography ensures the homogeneity of the protein polymerization state;finally,even after various conditions are optimized,the qualified protein crystals cannot be screened in prokaryotic expression.Using the Bac-to-Bac eukaryotic expression protein system which has a modification effect on the protein,the NPR1 protein is stably expressed by sf-9 cells,and the NPR1::pFastBacTM1 eukaryotic expression vector is constructed,and the vector is packaged into a virus pattern,ie,the bacmid is passed.The way the virus infects cells produces the protein of interest.The adherent cells were used to verify the target protein and expand the virus number.A large number of suspension cells were used to stably express the target protein,and the cells were lysed to purify the intracellularly released proteins,followed by ion exchange,gel filtration chromatography and a second crystal screening.In the screening kit,the crystal morphology was finally seen under the PEG/lon crystal screening conditions,and preliminary identification was performed using methylene blue staining.This thesis mainly uses two kinds of protein expression methods,and optimizes the protein conditions in each expression technology,according to the previous results to develop the corresponding optimization strategy to obtain as pure and high quality protein as possible,using crystal screening In the multiple crystal screening of the kit,the protein obtained by the currently used expression technology grows protein crystals.It is hoped that this will lay the foundation for the subsequent structural analysis.Through an in-depth understanding of the structure of the target protein,it hopes to propose new ideas for solving the problem in terms of structure,which will help in the later analysis of the function.