Study On The Response Mechanism Of Lactiplantibacillus Plantarum P9 To Phorate

With the exploration of the probiotic effects of lactic acid bacteria(LAB),the application of LAB is becoming more and more widespread,but it is often affected by various environmental stresses in practical applications.For example,agricultural workers will frequently exposed to various pesticides,and when such people take LAB,pesticide residues will inevitably have a stress effect on it and inhibit its cell activity.Phorate,a typical representative of organophosphorus pesticides,is widely used in crop production or landscape gardening construction because of its high efficiency,economy and broad spectrum.Therefore,we explored the response mechanism of LAB to pesticide stress by taking phorate as an example,which is a guideline for pesticide related workers to take LAB.Lactiplantibacillus plantarum P9,which has good probiotic properties and phorate degradation background,was selected as the target of this study.Based on the MRS culture system,samples of phorate-stressed were collected for metabolomics,transcriptomics and proteomics analysis.Key stress proteins were mined by association analysis,and prokaryotic expression and enzyme activity assays were performed to further explain the molecular mechanism of phorate stress.The main findings are as follows :(1)A total of 491 metabolites were identified based on widely targeted metabolomics analysis approach,and 231 differential metabolites were screened by a combination of univariate statistical analysis and multivariate statistical analysis,all of which were significantly up-regulated except for 2 metabolites that were significantly down-regulated.MSEA analysis revealed that they were mainly associated with 32 pathways including arginine biosynthesis,nitrogen metabolism,amyl-t RNA biosynthesis,valine,leucine and isoleucine biosynthesis,among which,arginine biosynthesis was the most responsive to phorate stress.It suggesting that this pathway is the main defense mechanism formed in Lactiplantibacillus plantarum P9.(2)A total of 3010 expressed genes were captured using the Illumina Hiseq sequencing platform,of which 64 differentially expressed genes were significantly up-regulated and 85 differentially expressed genes were significantly down-regulated.They affected 10 metabolic pathways including fatty acid biosynthesis,biotin metabolism,prodigiosin biosynthesis,alanine,aspartate and glutamate metabolism.20 differentially expressed genes were selected for q RT-PCR validation,and the results were consistent with the sequencing expression levels.(3)A total of 1400 proteins were identified by i TRAQ quantification and mass spectrometry,of which 47 differentially expressed proteins were significantly up-regulated and 45 differentially expressed proteins were significantly down-regulated.KEGG enrichment analysis revealed that they jointly regulate purine metabolic pathways,which are associated with cell proliferation.In addition,protein interaction network analysis revealed a synergistic mechanism of multiple stress proteins when stressed by phorate.(4)The protein sequences of key proteins PGPase and GLN were analyzed by online database and analysis software,and both proteins were subjected to prokaryotic expression and enzymatic activity were determined.The sequence analysis showed that PGPase is a phosphoglycolate phosphatase with a secondary structure consisting of 8 α-helices and 8β-chains,containing 4 key motifs,and a reasonable 3D structure could be predicted by homology modeling.GLN is a carbamoyl phosphate synthase with a secondary structure consisting of 8 α-helices and 17 β-chains,and a reasonable 3D structure was also obtained after homology modeling prediction.The results of enzymatic activity assay showed that although both proteins had some enzymatic activity,no phorate degradation activity was detected in the MRS medium.This indicates that the resistance of Lactiplantibacillus plantarum P9 to phorate stress is not through PGPase and GLN.In summary,the multi-omics study of Lactiplantibacillus plantarum P9 helps to uncover the molecular mechanism of its response to phorate stress,and in addition targeted prokaryotic expression is also an indispensable and important tool to further provide a theoretical basis for the application of LAB in pesticide stress.