Molecular Cloning And Expression Analysis Of A Full Length CDNA Peptidoglycan Recognition Protein S And L In Hyriopsis Cumingii

Pattern recognition receptors(PRRs) are innate immune molecules that recognize pathogen-associated molecular patterns(PAMPs) presented on microbial pathogens.Peptidoglycan recognition proteins(PGRPs) are a family of PRR that recognize bacterial peptidoglycan. PGRPs are highly conserved in invertebrates and vertebrates including Molluscs. In this study, the cDNA sequence of Hyriopsis cumingii PGRP-S(hcPGRP-S) and PGRP-L(hcPGRP-L) was cloned by 5′ and 3′ rapid amplification of cDNA ends methods(5′RACE, 3′RACE) based on two long gene sequence were determined by High-throughput transcriptome sequencing for transcriptome of H.cumingii. The tissue expression and the expression of these two genes in hepatopancreas after injected lipopolysaccharide or peptidoglycan was determined using quantitative real-time PCR(qRT-PCR).The sequence of hcPGRP-S cDNA was 1438 bp in length, consisting of a 858 bp open reading frame(ORF) encoding 285 amino acid residues with 32.3 ku of predicted molecular weight and 7.98 of the theoretical isoelectric point, which was predicted to have no signal petptide and transmembrane helices but found two latent N-glycosylation sites N40 PT and N92 YS. Multiple alignment analysis revealed that hcPGRP-S shares the highest identity with Euprymna scolopes PGRP4(51%).qRT-PCR demonstrated that the mRNA is constitutively expressed in all the examined organs of healthy H.cumingii, with the lower level in gill, heart and mantle,and with the higher in gonad and hepatopancreas. The expression of hcPGRP-S in hepatopancreas up-regulated obviously after injected 100 μl lipopolysaccharide(LPS,1ml/L) or 100 μl peptidoglycan(PGN, 1ml/L).The results indicated that hcPGRP-L cDNA sequence was 1716 bp in length,consisting of a 1263 bp open reading frame(ORF) encoding 420 amino acid residues with 48.24 ku of predicted molecular weight and 8.58 of the theoretical isoelectric point, which was predicted to have no signal petptide and transmembrane helices but found four latent N-glycosylation sites N67 QT, N99 KS, N193 LT and N229 SS, and found a PGRP domain(aa249-391). Multiple alignment analysis revealed that hcPGRP-L shares the identity between 53% to 62% with H.cumingii PGRP-1a,PGRP-S, PGRP-S1 and PGRP-S2. qRT-PCR demonstrated that the mRNA is constitutively expressed in all the examined organs of healthy H.cumingii, with the lower level in gill and heart, and with the higher in gonad and hepatopancreas. The expression of hcPGRP-L in hepatopancreas up-regulated obviously after injected peptidoglycan, but have no significant change after injected lipopolysaccharide.