Cloning, Expression And Structure Study Of Wheat Stripe Rust Effect Peotein Pst-2511 And Study On Palm Alkylation Modification Of 21 Peptides In PSD-95

Wheat stripe rust caused by wheat stripe rust(Puccinia striiformis f.sp.tritic)is a type of fungal disease that seriously damages wheat production in China,and the sterilizer is the only infective structure of wheat stripe rust,pathogen rust through the suction While acquiring nutrients from the host cells,a large number of effector proteins are secreted to the host cells by the stimulator to inhibit the host's defense response.However,the transport mechanism and pathogenesis of most of the effector proteins are still unclear.Because most of the effector proteins are difficult to find out from the amino acid sequence,it is difficult to study the function of rust effector proteins.In this context,protein structure biology has gradually become an important means for carrying out in-depth study of the effector protein function.Study on the structure and function of wheat stripe rust effector can provide theoretical basis for preventing wheat stripe rust.In this paper,the prokaryotic expression vector pET-32a-PP-pst-2511 of the wheat stripe rust effector gene pst-2511 was constructed and expressed in E.coli BL21(DE3).The expressed protein was used pro Purification by chromatography and high performance liquid chromatography yield soluble effector proteins.After identification by mass spectrometry,the molecular weight of the purified protein accords with the theoretical value.Circular dichroism spectra characterized the ?-helix of the secondary structure of the protein,and as the concentration of trifluoroethanol(TFE)increased,the ?-helix ratio increased.When the concentration of TFE was 30%,the results of circular dichroism analysis showed that the ?-helix and ?-sheet were similar to those predicted by bioinformatics.This study lays the foundation for the subsequent analysis of the three-dimensional structure of the effector proteins by NMR methods and their in vitro and in vivo protein interaction experiments.Post-synaptic density-95(PSD-95),a member of the membrane-associated guanylate kinase family,plays an irreplaceable role in the transmission of neural signals.Protein palmitoylation refers to an important form of covalent modification of lipids after protein translation.The main target is cytoplasmic proteins,which have multiple effects on protein function.Palmylated modification is also involved in a variety of cell biological processes andis closely related to the development of many diseases.The early stage of Alzheimer disease(AD)is characterized by memory impairment,which is closely related to the loss of synapse number and loss of synaptic function.Proline-rich PSD-95 and backbone protein Shank 1 are core structural proteins in postsynaptic dense regions and play an important role in maintaining normal function and plasticity of synapses.As a polypeptide in the PSD-95 protein,the 21 peptide has a certain degree of relation to the treatment of Alzheimer's disease by the product of its palm alkylation.The interaction between PSD-95 and CDKL-5 further shows that only PSD-95 in the palm alkylation state can play its role,and that its binding region with CDKL-5 is mainly N-terminal 1-21 Amino acids.In this study,the corresponding21 peptides were designed and synthesized.The 21 peptides were first reduced with a reducing solution containing DTT,purified by HPLC,and then lyophilized.The resulting powder was rapidly subjected to palmitic alkylation reaction.The palmitic alkylation is a click reaction triggered by UV light at 365 nm.Under the trigger of VA-044,the1-hexadecene double bond is opened on the thiol group of the 21 peptide.After the reaction,we also carried out HPLC purification and lyophilization..Change the water-soluble initiator VA-044 to oil-soluble AIBN.The occurrence of the reaction was confirmed by the gel electrophoresis gel diagram,and then through the mass spectrometry results,it was found that the molecular weight peak corresponding to the successful reaction appeared in the mass spectrum of the 21 peptide sample after the reaction,and the occurrence of the reaction was completely determined.The in vitro modification of the palmitoylation of the 21-mer peptide has been successful.The in vitro modification of the PSD-95 protein and the study of the mechanism of the PSD-95 in neurotransmission have important implications.At the same time,the palmitoylation site of 21 peptides can also be used as a drug target for research,which may provide a cure for diseases such as Alzheimer's disease and Reye's syndrome.