Gene Identification And Functional Analysis Of Peptidoglycan Recognition Protein From The Spotted Sea Bass(Lateolabrax Maculatus)

Peptidoglycan recognition protein?PGRPs?possess the function that the direct bactericidal effect and activate the host to initiate the immune-related signaling pathway,then synthesize antibacterial substanc?s which resist the invasion of external pathogens.The first PGRP was discovered in bombyx mori.According to the size of amino acid sequence,PGRPs of bombyx mori could be divided into two types,long and short,respectively.The PGRPs of bombyx mori could activate the phenol oxidase cascade reaction by recognizing the peptidoglycan in the cell wall of bacteria and specifically binding to it.Insect PGRPs mainly play the role of pattern recognition,signal transmission and effector molecules in the innate immune system.The human peptidoglycan recognition protein family includes four members,namely PGLYRP1,PGLYRP2,PGLYRP3 and PGLYRP4,which play the role of pattern recognition receptor and signal transmission.The PGRPs possessed the conserved PGRP domain at the C terminal,which was highly similar with the T7 lytic enzyme of bacteriophage,also named the type 2 n-acetylimyl-l alanine amidase,which can hydrolyze the peptidoglycan of bacterial cell wall.As an important link in the evolution of species,teleost fish has innate immunity and adaptive immune systems.However,due to the imperfection of its adaptive immune system,it mainly relies on the innate immune system in the process of resisting external pathogens.The innate immunity of teleost fish recognized the structure of pathogene-related pattern molecules which was expressed in pathogen organisms by PGRPs,and then activated the downstream signaling pathway to produce effector proteins or effector molecules for immune defense.At present,there are a total 23 of fish PGRPs reported.According to the amino acid sequence,PGRPs are mainly divided into three types,Short-PGRPs?PGRP-S?,Intermediate-PGRPs?PGRP-I?and Long-PGRPs?PGRP-L?,respectively.Studies had shown that PGRPs possessed the bactericidal activity and activate signaling pathways.In this study,the sea spotted bass?Lateolabrax maculatus?as one of the common marine fish species in China,was used as the material.We used the Illumina high-throughput sequencing technology to conduct transcriptome sequencing on the head kidney,liver and spleen from the sea spotted bass which was injection with Edwardsiella tarda?E.tarda?for 12 hours.A total of 22015 unigenes was detected by sequencing,and BLAST information was compared with Nr,GO,COG,KEGG and Swiss-Prot databases.The results showed that the highest rate of annotation was the Nr and KEGG database,95.68%and 96.73%,respectively.To analysis of the differentially expressed genes?DEGs??p-adjust<0.05?in groups which were injection with E.tarda by compared with the control groups.The results shown the 907?377 up,530 down?DEGs was found in head kidney,2542 DEGs?1302 up,1240 down?was found in liver and 1239 DEGs?726up,503 down?was found in spleen,respectively.Eight immune-related DEGs genes were randomly selected to verify by q RT-PCR,PGRP-SC2,PGRP2,IL-1?,LGP2,TLR5,TLR8,TLR13 and Hep,respectively.The results showed that the expression level of PGRP-SC2,PGRP2,IL-1?,TLR5,TLR8,TLR13 and Hep were consistent with the analysis results of RNA-seq,indicating that the results of the transcriptometer sequencing were reliable in this study.In this study,the gene sequences of PGRP2,PGPR-L2 and PGRP-SC2 were cloned by RT-PCR and RACE technology.The c DNA length of PGRP2,PGRP-L2 and PGRP-SC2 were 1583bp,2246bp and 1063bp,and the open read frame?ORF?were 1407bp,1449bp and 558bp,the amino acid sequences encoded were 468,482 and 167 aa,respectively.PGRP2 and PGRP-L2 possess the signal peptides at the N terminal,with 24and 21 amino acid residues,respectively,while PGRP-SC2 has no signal peptides.Previous studies showed that PGRP-S and PGRP-L from the different species evolved independently.It was means that the PGRPs from the same type clustered into a cluster,while the homology with other species was relatively low,which was consistent with the result of evolutionary tree analysis.Through homologous analysis of amino acids,it can be found that their c-terminus all PGRPS possessed the PGRP domain at C-terminus,which was also named the type II n-acetyl-l-alanine amidase.This domain also including the amino acid sites bound to zinc ions,which constitute with 2 His,1 Tyr and 1 Cys.The results shown that PGRP-L2,PGRP2 and PGRP-SC2 of the sea spotted bass had high homology with members of the PGRPs superfamily from other teleost fish by comparative analysis of evolutionary tree and amino acid homology,indicating that PGRP-L2,PGRP2 and PGRP-SC2 of the sea spotted bass as the important members of the peptidoglycan recognition family in teleost fish.The tissue distribution of PGRPs and the gene expression after external pathogen stimulation were detected by quantitative real-time PCR.The results showed that PGRPs m RNA were distributed in all examined tissues.In particular,PGRP2 gene was mainly expressed in the spleen and gill,PGRP-L2 gene was strongly expressed in the liver,and PGRP-SC2 gene was strongly expressed in the gill,indicating that the sea spotted bass could rely on different PGRPs to activate the downstream signaling pathway and activate the immune system to resist the invasion of external pathogens.The sea spotted bass injection with LPS,PGRPs gene were significantly up-regulated in all examined tissues which including the head kidney,spleen,intestine and gill.In detail,PGRP2 and PGRP-L2 gene significantly up-regulated in the head kidney and spleen,PGRP-SC2 gene significantly strong expressed in the intestinal,however,the expression of PGRPs in different tissue were different,indicating that the sea spotted bass PGRPs genes play the different role in against gram-negative bacteria invasion.After injection with Poly?I:C?,PGRPs gene was significantly up-regulated in all the tested tissues,indicating that PGRPs also played an important role in resisting virus invasion.And the PGRP-SC2 gene was significantly stronger expressed than PGRP2 and PGRP-L2 genes,indicating that PGRP-SC2 gene may play a more important role in resisting virus stimulation.After infection with E.tarda,the expression of PGRPs gene were significantly up-regulated in each tissue and the expression of PGRP-SC2 and PGRP-L2 gene was obviously stronger than PGRP2 gene,moreover,the expression of the PGRP-SC2 and PGRP-L2 injection with E.tarda was stronger than injection with LPS,Poly?I:C?,may be LPS,Poly?I:C?as the analogues of gram-negative bacteria and viruses,respectively,and E.tarda was the living bacterium so PGRPs gene expression were more intense.Through simple biochemical experiments,it was proved that PGRP2,PGRP-L2 and PGRP-SC2 of the sea spotted bass possessed the amidase activity,and the amidase activity was dependent on Zn²⁺,moreover,the amidase activity of PGRP2 was significantly stronger than PGRP-L2 and PGRP-SC2.The PGRPs recombinant protein showed the inhibitory effect to growth curve of Vibrio harveyi,E.tarda and Staphylococcus aureus,and the higher the concentration of the protein inhibiting effect was more obvious,however,its inhibition depends on the Zn²⁺,indicating that sea spotted bass PGRPs recombinant protein has a broad spectrum bactericidal and the inhibition may be related to amide enzyme activity.The plate perforation experiment further confirmed that PGRPs protein has bactericidal effect,but it did not possess the direct bactericidal effect which was dependent on Zn²⁺.Finally,the proteins were combined in pairs to verify whether the PGRPs proteins have superposition effect on different bacteria.The results showed that PGRPs possessed the different bactericidal effects on different bacterial,and PGRPs protein mixed in pairs showed significantly better bactericidal effects than single PGRPs protein,indicating that PGRPs not only played the role of pattern recognition and amidase activity,but also showed the synergistic superposition effect in the process of resisting external pathogens.