Cloning Of The Peptidoglycan Recognition Protein PGRP-S2 From Plutella Xylostella And Analysis Of Its Microbial Binding Function

Plutella xyostella is a member of the family Lepidoptera,which is one of the most destructive pest of cruciferous crops such as broccoli,cabbage,and radish.As a pathogen recognition protein,peptidoglycan recognition protein(PGRP)plays an important role in the resistance of the P.xylostella to pathogenic microorganisms.In order to explore the mechanism of peptidoglycan recognition protein and its expression regulation mechanism,a peptidoglycan recognition protein PGRP-S2 was cloned and identified in our study using the diamondback moth as an experimental material,and the mechanism of action and expression of PGRP-S2 was elucidated.The main results were as follows:1.According to the genomic information of diamondback moth,the open reading frame(ORF)of PGRP-S2 in the diamondback moth P.xylostella was cloned by RACE.The bioinformatics analysis showed that the protein encoded by the sequence contained signal peptide,PGRP domain and amidase domain structure,this indicated that the protein may have the function of recognition and degradation bacterial peptidoglycan,which plays an important role in resistance to pathogenic microorganisms in diamondback moth.2.The target protein PGRP-S2 was successfully obtained by using prokaryotic expression system and AKTA protein purification system.Western blot analysis showed that PGRP-S2 protein could be combined with Escherichia coli and Staphylococcus aureus;the results of agglutination test showed that,compared with the control group,the cell agglutination occurred in E.coli and S.aureus,which were treated by PGRP-S2 protein;the results of antibacterial test showed that the effect of PGRP-S2 on E.coli and S.aureus had no significant antibacterial activity.The above experiments showed that PGRP-S2 protein has the function of binding and identifying with bacteria,but did not directly kill the bacteria,the mechanism of action may be through the recognition of peptidoglycan and activation of a series of downstream signal pathways to produce antimicrobial peptides to resist pathogens.3.According to the genomic information of the diamondback moth P.xylostella,the PGRP-S2 5’ flanking sequence PGRP-S2-P-1937--1 was cloned.The results of BDGP analysis showed that the four regions of the sequences of PGRP-S2-P-1642--1593,PGRP-S2-P-1254--1505,PGRP-S2-P-714--655 and PGRP-S2-P.160--111 may be transcription initiation regions;search for CARE analysis showed that the flanking sequence contains a lot of TATA-box and CAAT-box sequences.According to the results of bioinformatics analysis,the flanking sequence of PGRP-S2-P-1937--1 was subcloned and the expression vector pET28a-PGRP-S2-P-x-GFP of the 5’-end deletion fragment of the P.xylostella PGRP-S2 gene was constructed using GFP as a reporter gene and the flanking sequence functional identification strain was identified by fluorescence microscopy.The results showed that the flanking sequence of PGRP-S2-P-1937--1285,PGRP-S2-P-1467--1078 and PGRP-S2-P-986--518 was able to initiate GFP expression in E.coli with function of promoter activity;the sequence of-1937～-1492 is gene encoding Acromyrmex echinatior histone-lysine N-methyltransferase SETMAR,showed that the PGRP-S2 gene 5’ flanking sequence of the-1937～-1285 is not PGRP-S2 gene promoter;the fluorescence intensity indicated that the promoter activity of sequence PGRP-S2-P-986--518 was stronger than that of sequence PGRP-S2-P-1937--1285,suggesting that the promoter region of PGRP-S2 gene existed mainly in-986～-518 bp region.While the functional activity of the promoters of the sequences PGRP-S2-P-1937--1、PGRP-S2-P-1467--1,PGRP-S2-P-986--1,PGRP-S2-P-526--1 and PGRP-S2-P-205--1 were very weak,it is indicated that the transcriptional regulation of PGRP-S2 gene promoter was negatively regulated in the-205～-1 bp region.Peptidoglycan recognition protein plays an important role in the process of resistance to pathogenic microorganisms of the diamondback moth P.xylostella.Based on the in vitro protein expression technology and the interaction relationship between recombinant protein PGRP-S2 and microbial pathogens E.coli and S.aureus,the mechanism of action of peptidoglycan recognition protein PGRP-S2 was studied in depth;at the same time,the expression regulation mechanism of peptidoglycan recognition protein PGRP-S2 was studied by the functional identification of the fragment of the 5’ flanking sequence.The immune mechanism of the P.xylostella against bacterial infection was initiated by the immune system against the pathogenic microorganisms of the P.xylostella.It provides a theoretical basis for the development of new bacterial insecticides.