Molecular cloning of the heat shock protein 20 gene from Paphia textile and its expression in response to heat shock

P. textile is an important aquaculture species in China and is mainly distributed in Fujian, Guangdong, and Guangxi Provinces. In this study, an HSP20 c DNA designated Pt HSP20 was cloned from P. textile. The full-length c DNA of Pt HSP20 is 1 090 bp long and contains a 5′ untranslated region(UTR) of 93 bp, a 3′ UTR of 475 bp, and an open reading frame(ORF) of 522 bp. The Pt HSP20 c DNA encodes 173 amino acid residues and has a molecular mass of 20.22 k Da and an isoelectric point of 6.2. Its predicted amino acid sequence shows that Pt HSP20 contains a typical α-crystallin domain(residues 77–171) and three polyadenylation signal-sequences at the C-terminus. According to an amino acid sequence alignment, Pt HSP20 shows moderate homology to other mollusk s HSPs. Pt HSP20 m RNA was present in all of the test tissues including the heart, digestive gland, adductor muscle, gonad, gill, and mantle, with the highest concentration found in the gonad. Under the stress of high temperature, the expression of Pt HSP20 m RNA was down-regulated in all of the tissues except the adductor muscle and gonad.

Molecular cloning, characterization and expression analysis of a catalase gene in Paphia textile

Catalase is an important antioxidant protein that can protect organisms against various forms of oxidative damage by eliminating hydrogen peroxide. In this study, the catalase c DNA of Paphia textile（Pt CAT） was cloned using RTPCR and rapid amplification of c DNA ends（RACE）. Pt CAT is 1 921 bp long and consists of a 5′-UTR of 50 bp, a 3′-UTR of 349 bp, and an ORF of 1 542 bp that encodes 513 amino acids with a molecular weight of 58.4 k D and an estimated isoelectric point of 8.2. Sequence alignment indicated that Pt CAT contained a highly conserved catalytic signature motif（^（61）FNRERIPERVVHAKGAG^（77））, a proximal heme-ligand signature sequence（^（352）RLFSYSDP^（359））, and three catalytic amino acid residues（H^（72）, N^（145）, and Y^（356））. Pt CAT also contains two putative N-glycosylation sites（^（34）NKT^（36） and ^（437）NFT^（439）） and a peroxisome-targeting signal（^（511）AQL^（513））. Furthermore, Pt CAT shares 53%–88% identity and 29%–89% similarity with other catalase amino acid sequences. Pt CAT m RNA was present in all tested organs, including the heart, digestive gland, adductor muscle, gonad, gill, and mantle, but its expression was highest in the digestive gland. High-temperature-induced stress produced two expression patterns of Pt CAT m RNA： first, an initial up-regulation followed by a down-regulation in the heart, digestive gland, and gonad and, second, consistent down-regulation in all other organs. These results demonstrate that Pt CAT is a typical member of the catalase family and might be involved in the responses to harmful environmental factors.

Isolation and identification of pigment substances in orange feet of Paphia textile

Paphia textile is an economically important bivalve cultured in southeastern China.The feet,as the main edible part of P.textile,vary in color from white to orange.Previous studies have suggested that the orange feet of P.textile contain higher levels of carotenoids than the white feet,but the kinds of carotenoids are not known.The pigments identified in this study can facilitate further analysis of their underlying molecular mechanism and enable the breeding of P.textile with high carotenoid content.The pigments were extracted from the orange feet of P.textile by an acetone extraction method,and then,the extracted pigments were analyzed by liquid chromatography-mass spectrometry(LC-MS).Moreover,the MS analysis was carried out with an electrospray ionization(ESI)source in the positive(ESI+)and negative(ESI-)ion modes.A total of 47 substances were detected,including 9 terpenoids,7 amino acids and peptides,13 lipids,9 organic acids,2 types of vitamin A and 7 other substances.Furthermore,β-doradecin(3’-hydroxy-3,4-diketo-β-carotene)was identified as the main pigment in the feet.This is the first report of the presence ofβ-doradecin,a type of carotenoid,in bivalves.