Molecular characterization, expression and immune responses to lipopolysaccharide and peptidoglycan stimulation of Se-GPx gene in Manila clam Ruditapes philippinarum
WANG Jun1,2， LIANG Teng1, WANG Huamin1, YAN Xiwu1,2，DING Jianfeng1,2*
1.College of Fisheries and Life Science, Dalian Ocean University, Dalian 116023, China; 2. Engineering Research Center of Shellfish Culture and Breeding in Liaoning Province, Dalian 116023, China
Abstract: The selenium-dependent glutathione peroxidase (Se-GPx) gene (designated as Se-GPx-a and Se-GPx-b was cloned and expressed in adductor muscle, mantle, gill, water tube, gonad, hepatopancreas, and feet of orange, white and zebrawhite Manila clam Ruditapesphilippinarum with shell length of (2.0±0.5)cm injected hemolymphically with lipopolysaccharide solution(LPS)and peptidoglycan solution (PGN) of 100 μg/mL at a dose of 50 μL per clam at water temperature of (18.0±0.5)℃ by RACE technology and RT-PCR method to investigate the molecular characterization, expression and immune responses to lipopolysaccharide and peptidoglycan stimulation of selenium- dependent Se-GPx gene in Manila clam. It was found that the full-length cDNA of Se-GPx-a and Se-GPx-b had the open reading frame (ORF) of 582 bp in the RpSe-GPx-a and 633 bp in RpSe-GPx-b, encoding two polypeptides of 193 and 210 amino acids in length. There were conserved selenocysteine insertion sequence (SECIS) in the 3′untranslated region (UTR) of the RpSe-GPx showing all the conserved features critical for the fundamental structure and function of glutathione peroxidase, including the selenocysteine encoded by stop codon UGA, the GPx signature motif (GKVVLVENVASLUGTT) and the active site motif (WNFEKF), with high homology of RpSe-GPx with other mollusc species. Tissue expression analysis revealed that there was the maximal expression level of RpSe-GPx in the hepatopancreas (P<0.05), followed by the mantle and gills, and the minimal expression level in the adductor muscle (P<0.05). The maximal expression level of Se-GPx-a and Se-GPx-b mRNA in Manila clam injected hemolymphically with LPS were detected in the white-shell color group at 12 and 6 h, and in the white zebra-shell color group at 48 h and 6 h, and in the orange-shell color group at 3 h and 24 h(P<0.05). The maximal expression level of Se-GPx-a and Se-GPx-b in Manila clam injected hemolymphically with PGN were all significantly induced at 24 h in white-shell color group, and at 24 h in the white zebra-shell color group, and at 48 h in the orange-shell color group(P<0.05). The findings indicated that different responses of the RpSe-GPx gene to LPS and PGN stimulation were attributed in the difference in the melanin formation process in the three shell-color clam, which provided data for shell color selection breeding and healthy farming of Manila clam.
2021, Vol. 36 
