Identification and functional characterization of a superoxide dismutase (CuZnSOD) from Pinctada fucata martensii.

Copper/zinc superoxide dismutase (Cu/Zn-SOD) can effectively eliminate reactive oxygen species (ROS)，avoid damage from O2 to the body, and maintain O2 balance. In this study, multi-step high-performance liquid chromatography (HPLC), combined with Mass Spectrometry (MS), was used to isolate and identify Cu/Zn-SOD from the serum of Pinctada fucata martensii (P. f. martensii) and was designated as PmECSOD. With a length of 1864 bp and an open reading frame (ORF) of 1422 bp, the cDNA encodes a 473 amino acid protein. The PmECSOD transcript was detected in multiple tissues by quantitative real-time PCR (qRT-PCR), with its highest expression level being in the gills. Additionally, the temporal expression of PmECSOD mRNA in the hemolymph was highest at 48 h after in vivo stimulation with Escherichia coli and Micrococcus luteus. The results from this study provide a valuable base for further exploration of molluscan innate immunity and immune response.

Mitf Involved in Innate Immunity by Activating Tyrosinase-Mediated Melanin Synthesis in Pteria penguin.

The microphthalmia-associated transcription factor (MITF) is an important transcription factor that plays a key role in melanogenesis, cell proliferation, survival and immune defense in vertebrate. However, its function and function mechanism in bivalve are still rarely known. In this research, first, a Mitf gene was characterized from Pteria penguin (P. penguin). The PpMitf contained an open reading frame of 1,350 bp, encoding a peptide of 449 deduced amino acids with a highly conserved basic helix-loop-helix-leucine zipper (bHLH-LZ) domain. The PpMITF shared 55.7% identity with amino acid sequence of Crassostrea gigas (C. gigas). Tissue distribution analysis revealed that PpMitf was highly expressed in mantle and hemocytes, which were important tissues for color formation and innate immunity. Second, the functions of PpMitf in melanin synthesis and innate immunity were identified. The PpMitf silencing significantly decreased the tyrosinase activity and melanin content, indicating PpMitf involved in melanin synthesis of P. penguin. Meanwhile, the PpMitf silencing clearly down-regulated the expression of PpBcl2 (B cell lymphoma/leukemia-2 gene) and antibacterial activity of hemolymph supernatant, indicating that PpMitf involved in innate immunity of P. penguin. Third, the function mechanism of PpMitf in immunity was analyzed. The promoter sequence analysis of tyrosinase (Tyr) revealed two highly conserved E-box elements, which were specifically recognized by HLH-LZ of MITF. The luciferase activities analysis showed that Mitf could activate the E-box in Tyr promoter through highly conserved bHLH-LZ domain, and demonstrated that PpMitf involved in melanin synthesis and innate immunity by regulating tyrosinase expression. Finally, melanin from P. penguin, the final production of Mitf-Tyr-melanin pathway, was confirmed to have direct antibacterial activity. The results collectively demonstrated that PpMitf played a key role in innate immunity through activating tyrosinase-mediated melanin synthesis in P. penguin.

Cloning, characterization, and functional analysis of chitinase-like protein 1 in the shell of Pinctada fucata.

Biomineralization, especially shell formation, is a sophisticated process regulated by various matrix proteins. Pinctada fucata chitinase-like protein 1 (Pf-Clp1), which belongs to the GH18 family, was discovered by our group using in-depth proteomic analysis. However, its function is still unclear. In this study, we first obtained the full-length cDNA sequence of Pf-Clp1 by RACE. Real-time polymerase chain reaction results revealed that Pf-Clp1 was highly expressed in the important biomineralization tissues, the mantle edge and the mantle pallial. We expressed and purified recombinant protein rPf-Clp1 in vitro to investigate the function of Pf-Clp1 on CaCO3 crystallization. Scanning electron microscopy imaging and Raman spectroscopy revealed that rPf-Clp1 was able to affect the morphologies of calcite crystal in vitro. Shell notching experiments suggested that Pf-Clp1 might function as a negative regulator during shell formation in vivo. Knockdown of Pf-Clp1 by RNAi led to the overgrowth of aragonite tablets, further confirming its potential negative regulation on biomineralization, especially in the nacreous layer. Our work revealed the potential function of molluscan Clp in shell biomineralization for the first time and unveiled some new understandings toward the molecular mechanism of shell formation.

Survival, retention rate and immunity of the black shell colored stocks of pearl oyster Pinctada fucata martensii after grafting operation.

We have developed a black shell colored selected line observed to have higher survival ability. In this study, to understand its immune capacity, total carotenoid content (TCC) of the black shell colored line (BG) and the control group (CG) were compared. Survival and retention rates, immunity and antioxidant capacity of BG were compared relative to CG at different times after grafting operation. The results showed that BG had significantly larger TCC than CG (P < 0.05). BG had significantly higher survival and retention rates than CG on days 7, 30 and 360 after grafting (P < 0.05). On days 360, BG had significantly larger pearl thickness than CG (P < 0.05). BG exhibited increased ACP, AKP, SOD, CAT, TAOC and LZ activity than the CG on 0 h, 12 h, 1 d, 3 d, 5 d, 7 d and 30 d after grafting. BG had higher expression levels of Fascin, SOD, CDK-7, CDAP-1, IRAK-1, α2m, GST-1, TRAF-3 and Caspase-2 than CG. The results suggested that BG had higher immune competence and pearl production performances, which is promising to improve pearl quality and production.

Molecular and functional analysis of PmCHST1b in nacre formation of Pinctada fucata martensii.

Keratan sulfate possesses considerable amounts of negatively charged sulfonic acid groups and participates in biomineralization. In the present study, we investigated characteristics and functions of a CHST1 gene identified from the pearl oyster Pinctada fucata martensii (PmCHST1b) which participated in the synthesis of keratan sulfate. PmCHST1b amino acid sequence carried a typical sulfotransferase-3 domain (sulfotransfer-3 domain) and belonged to membrane-associated sulfotransferases. Homologous analysis of CHST1 from different species showed the conserved motif (5' PSB motif and 3' PB motif) which interacted with 3'-phosphoadenosine-5'-phosphosulfate (PAPS). Structure analysis of sulfotransferase domain indicted that PmCHST1b showed the conserved catalytic structure character and the relationships presented in the phylogenetic tree conformed to that of traditional taxonomy. Expression pattern of PmCHST1b in different tissues and development stages showed that PmCHST1b widely expressed in all the detected tissues and development stages and showed the highest expression level in the central zone of mantle (MC). PmCHST1b expressed highly in the trochophore, D-stage larvae and spat which corresponded to prodissoconch and dissoconch shell formation, respectively. RNA interference (RNAi) successfully inhibited expression level of PmCHST1b in MC (P<0.05), and sulfate polymer content in the extrapallial fluid significantly reduced (P<0.05). Crystallization of shell nacre became irregular. Results above indicated that PmCHST1b may affect nacre formation by participating in synthesis of keratan sulfate in extrapallial fluid. This study provided fundamental materials for further research on the role of sulfotransferases and keratan sulfate in nacre formation.

Serum immune response of pearl oyster Pinctada fucata to xenografts and allografts.

The mantle piece from the donor pearl oyster would be rejected by the immune system of recipient oyster in pearl culture practice, especially in the case that the donor and receptor are different species. Thus, investigation of the immune response of recipient oyster to grafted mantle pieces, particularly to xenografts, is of importance in creating xenograft transplantation technology for pearl culture industry. The humoral immune responses of P. fucata to allograft (mantle piece of P. fucata) and xenografts (mantle pieces of P. maxima and P. margaritifera, respectively) were studied in this paper. The oysters receiving no transplantations were served as the control group. The serum was collected from recipient P. fucata at 1 d, 2 d, 3 d, 4 d, 5 d, 7 d, 9 d, 11 d, 13 d, and 15 d, respectively after transplantation, and the serum antibacterial activity, lysozyme activity (LZM), alkaline phosphatase (AKP), acid phosphatase (ACP), total antioxidant capacity (TAC), and agglutination to rabbit red blood cells were investigated. The result indicated that serum of both the experimental groups and the control group can agglutinate rabbit red blood cells, with variation between groups and between time points, respectively. The antibacterial activity in the experimental group was significantly higher than that in the control group at 2-4 d, but lower at 5-11 d and returned back to normal at 15 d, with significant differences among experimental groups (P < 0.05). The LZM in the experimental group was significantly higher than that in the control group at 3-7 d, with significant differences in bacteriolytic activity among various groups (P < 0.05). Both the ACP and AKP activity levels in the experimental groups were higher than those in the control group at 2-9 d, with significant differences among various groups at 3-9 d (P < 0.05). The TAC level in the experimental groups was higher than that in the control group at 1-7 d, with significant differences among various groups at 4-7 d (P < 0.05). The above results indicated that all of the humoral immune factors investigated showed immune responses to both allografts and xenografts, with no specific to any of them. Thus, it is necessary to further screen immune rejection factors specific to xenografts, including cellular immune components.

Molecular characterization and expression analysis of chitinase from the pearl oyster Pinctada fucata.

Chitinase is an enzyme that plays an important role in the chitin metabolism of a wide range of organisms. However, the function of chitinase in the pearl oyster Pinctada fucata is yet to be determined. In this study, a chitinase gene (named PfChi1) was cloned from P. fucata and its expression profiles were investigated. The full-length cDNA of PfChi1 was 2743bp and consisted of a 2187-bp open reading frame encoding 728 amino acid residues, a 47-bp 5'-untranslated region (UTR), and a 509-bp 3'-UTR. Similar to other known chitinases, the PfChi1 protein is composed of an N-terminal leading signal peptide, a catalytic domain, a linker region, and a C-terminal chitin-binding domain. The results of qRT-PCR showed that PfChi1 was expressed in a wide range of tissues with relatively high levels in the mantle, muscle, gill, and gonad, and relatively low levels in hemocytes, the intestine, and the digestive gland (P<0.05). In situ hybridization showed that PfChi1 was mainly expressed in the mantle edge, particularly in the outer epithelial cells of the inner fold, whereas few hybridization signals were detected in the inner epithelial cells of the middle fold. A shell damage experiment indicated that PfChi1 transcript levels were up-regulated significantly (P<0.05) at 24h after shell damage and decreased gradually thereafter, followed by shell regeneration, indicating that PfChi1 is involved in shell formation. In addition, PfChi1 expression was higher in trochophore larvae than in other developmental stages (P<0.05), indicating a possible association with the formation of prodissoconch shells. To the best of our knowledge, this study is the first to report the potential biomineralization function of a chitinase in P. fucata.

Genomic structure of the α-amylase gene in the pearl oyster Pinctada fucata and its expression in response to salinity and food concentration.

Amylase is one of the most important digestive enzymes for phytophagous animals. In this study, the cDNA, genomic DNA, and promoter region of the α-amylase gene of the pearl oyster Pinctada fucata were cloned by using reverse transcription-polymerase chain reaction (RT-PCR), rapid amplification of cDNA ends, and genome-walking methods. The full-length cDNA sequence was 1704bp long and consisted of a 5'-untranslated region of 17bp, a 3'-untranslated region of 118bp, and a 1569-bp open reading frame encoding a 522-aa polypeptide with a 20-aa signal peptide. Sequence alignment revealed that P. fucata α-amylase (Pfamy) shared the highest identity (91.6%) with Pinctada maxima. The phylogenetic tree showed that it was closely related to P. maxima, based on the amino acid sequences. The genomic DNA was 10850bp and contained nine exons, eight introns, and a promoter region of 3932bp. Several transcriptional factors such as GATA-1, AP-1, and SP1 were predicted in the promoter region. Quantitative RT-PCR assay indicated that the relative expression level of Pfamy was significantly higher in the digestive gland than in other tissues (gonad, gills, muscle, and mantle) (P<0.001). The expression level at salinity 27‰ was significantly higher than that at other salinities (P<0.05). Expression reached a minimum when the algal food concentration was 16×10(4)cells/mL, which was significantly lower than the level observed at 8×10(4)cells/mL and 20×10(4) cells/mL (P<0.05). Our findings provide a genetic basis for further research on Pfamy activity and will facilitate studies on the growth mechanisms and genetic improvement of the pearl oyster P. fucata.

Molecular characterization and expression analysis of purple acid phosphatase gene from pearl oyster Pinctada martensii.

Purple acid phosphatases (PAPs), also known as type 5 acid phosphatases, are widely present in animals, plants, and fungi. In mammal, PAP was reported to participate in immune defense and bone resorption. In this study, the characteristics and potential functions of a PAP gene from pearl oyster Pinctada martensii (pm-PAP) were examined. The Pm-PAP cDNA was found to be 2777 base pairs, containing a 1581-base pair open reading fragment encoding for 526 amino acids with an estimated molecular mass of 60.1 kDa and theoretical isoelectric point of 5.82. One signal peptide and five conserved motifs [GDXX/GDXXY/GNH(D/E)/XXXH/(A/G)HXH] were present in the entire sequence. Tissue expression profile analysis showed that pm-PAP mRNA was constitutively expressed in all tissues studied with abundant mRNA found in mollusk defense system, including hepatopancreas, gill, and hemocytes. After lipopolysaccharide stimulation, the expression of pm-PAP mRNA in hemocytes was dramatically upregulated at 2 h and achieved the highest level at 36 h. Additionally, pm-PAP mRNA expression was significantly increased and achieved the highest level at 2 days after the surgical implantation during pearl production. These results suggest that pm-PAP is a constitutive and inducible protein that may be involved in the immune defense of pearl oyster.

Evolution of the tyrosinase gene family in bivalve molluscs: independent expansion of the mantle gene repertoire.

Tyrosinase is a copper-containing enzyme that mediates the hydroxylation of monophenols and oxidation of o-diphenols to o-quinones. This enzyme is involved in a variety of biological processes, including pigment production, innate immunity, wound healing, and exoskeleton fabrication and hardening (e.g. arthropod skeleton and mollusc shell). Here we show that the tyrosinase gene family has undergone large expansions in pearl oysters (Pinctada spp.) and the Pacific oyster (Crassostrea gigas). Phylogenetic analysis reveals that pearl oysters possess at least four tyrosinase genes that are not present in the Pacific oyster. Likewise, C. gigas has multiple tyrosinase genes that are not orthologous to the Pinctada genes, indicating that this gene family has expanded independently in these bivalve lineages. Many of the tyrosinase genes in these bivalves are expressed at relatively high levels in the mantle, the organ responsible for shell fabrication. Detailed comparisons of tyrosinase gene expression in different regions of the mantle in two closely related pearl oysters, P. maxima and P. margaritifera, reveals that recently evolved orthologous tyrosinase genes can have markedly different expression profiles. The expansion of tyrosinase genes in these oysters and their co-option into the mantle's gene regulatory network is consistent with mollusc shell formation being underpinned by a rapidly evolving transcriptome.

Genomic structure, characterization and expression analysis of a manganese superoxide dismutase from pearl oyster Pinctada fucata.

Manganese superoxide dismutase (MnSOD) is a major component of the cellular defense mechanisms against oxidative damage. We cloned and analyzed the expression pattern and genomic structure of the MnSOD gene of pearl oyster Pinctada fucata, hereafter designated as PoMnSOD. The full-length PoMnSOD cDNA was 1080 bp in length and consisted of a 5'-untranslated region (UTR) of 222 bp, a 3'-UTR of 318 bp with a polyadenylation signal (AATAAA) at 15 nucleotides upstream of the poly (A) tail, and an open reading frame (ORF) of 540 bp encoding a polypeptide of 180 amino acids with an estimated molecular mass of 20.4 kDa and a predicted pI of 6.72. Sequence analysis showed that PoMnSOD contained MnSOD family signatures F(44)NGGGHLNH(52), I(97)QGSGWGWLA(106) and D(138)VWEHAYY(145), four conserved residues for manganese metal binding (H(4), H(52), D(138) and H(142)), and two potential N-glycosylation sites (N(33) and N(51)). Homology analysis revealed that PoMnSOD shared 47.6-55.9% identity and 57.4-65.6% similarity to the other known PoMnSOD amino acid sequences. PoMnSOD genomic DNA was 5040 bp in length and contained three exons and two introns, which was a tripartite organization and coincided with the consensus GT-AG splicing rule. PoMnSOD promoter contained the various transcription factors associated with the immune modulation and stress responses. Quantitative RT-PCR analysis demonstrated that PoMnSOD was constitutively expressed in all detected tissues, and PoMnSOD mRNA expression was significantly up-regulated in intestine, mantle, gills, digestive gland and haemocytes after Vibrio alginolyticus injection. These results suggested that PoMoSOD was an acute-response protein involved in the innate immune responses of pearl oyster, and provided general information about the mechanisms of innate immune defense against bacterial infection in pearl oyster.

Molecular cloning, characterization and expression analysis of cytoplasmic Cu/Zn-superoxide dismutase (SOD) from pearl oyster Pinctada fucata.

Because of its capacity to rapidly convert superoxide to hydrogen peroxide, superoxide dismutase (SOD) is crucial in both intracellular signalling and regulation of oxidative stress. In this paper we report the cloning of a Cu/Zn SOD (designated as pfSOD) from the pearl oyster (Pinctada fucata) using rapid amplification of cDNA ends (RACE) PCR. The full-length cDNA of this Cu/Zn SOD contains an open reading frame (ORF) of 471 bp coding for 156 amino acids. No signal peptide was identified at the N-terminal amino acid sequence of Cu/Zn SOD indicating that this pfSOD encodes a cytoplasmic Cu/Zn SOD. This is supported by the presence of conserved amino acids required for binding copper and zinc. Semi-quantitative analysis in adult tissues showed that the pfSOD mRNA was abundantly expressed in haemocytes and gill and scarcely expressed in other tissues tested. After challenge with lipopolysaccharide (LPS), expression of pfSOD mRNA in haemocytes was increased, reaching the highest level at 8 h, then dropping to basal levels at 36 h. These results suggest that Cu/Zn SOD might be used as a bioindicator of the aquatic environmental pollution and cellular stress in pearl oyster.

Molecular cloning and characterization of the 5'-flanking regulatory region of the carbonic anhydrase nacrein gene of the pearl oyster Pinctada fucata and its expression.

The carbonic anhydrase nacrein participates in the formation of the nacreous or prismatic layer of Pinctada fucata. We isolated a genomic clone containing the nacrein gene and cloned the 5'-flanking region. Within the 1336 bp 5' flanking region, we identified putative cis-acting elements, including the TATA box (TATAAAA) at -82 bp, and AP1 (-819 bp) and Oct-1 (-1244 bp) binding sites. In addition to the mantle, the nacrein gene is also expressed in the adductor muscle, liver, and foot. These results showed that nacrein not only takes part in the formation of the hard tissue but also might be involved in acid-base balance, ion transport, and maintenance of ionic concentration. In vitro transcription experiments showed that the addition of human c-jun activates transcription from the nacrein promoter. This is the first report of a promoter from a gene that controls the formation of the hard tissue of mollusk shells.

Sequence of the pearl oyster carbonic anhydrase-related protein and its evolutionary implications.

Carbonic anhydrases are conserved in vertebrates and invertebrates, and a noncatalytic carbonic anhydrase-related protein VIII (CARP VIII) has been found in deuterostomes and the phylum Placozoa. I isolated a cDNA encoding a noncatalytic CARP from the mantle of the pearl oyster Pinctada fucata. The polypeptide (CARP-1) predicted from the nucleotide sequence shares 44-60% identity with known CARP VIII sequences, and its phylogenetic analysis showed that P. fucata formed a single group with deuterostome invertebrates. However, since CARP VIII sequences are not identified in protostomes, these results suggest that CARP-1 may have originated in molluscs independently from deuterostome CARP VIII sequences.

Molecular characterization and mRNA expression of catalase from pearl oyster Pinctada fucata.

Catalase (EC 1.11.1.6) is an important antioxidant enzyme that protects aerobic organisms against oxidative damage by degrading hydrogen peroxide to water and oxygen. In the present study, a catalase cDNA of peal oyster Pincatada fucata (designated as PoCAT) is cloned and characterized by expressed sequence tag (EST) and rapid amplification of cDNA ends (RACE) methods. PoCAT is 2428 bp long and consists of a 5'-UTR of 140 bp, an unusually long 3'-UTR of 749 bp, and an open reading frame (ORF) of 1539 bp. The ORF of PoCAT encodes a polypeptide of 512 amino acids with molecular weight of 58.1 kDa and the theoretical isoelectric point of 8.4. PoCAT shares 62.3-82.2% identity and 73.0-92.0% similarity to other catalase amino acid sequences. Sequence alignment indicates that PoCAT contains the proximal heme-ligand signature sequence (R³5¹LFSYSDT³58), the proximal active site signature (F6¹NRERIPERVVHAKGGGA78), and the three catalytic amino acid residues (His7², Asn¹45, and Tyr³55). PoCAT has two potential glycosylation sites (N4³6YS4³8 and N478FS48°) and a peroxisome targeting signal (ASL). PoCAT mRNA was ubiquitously expressed in all detected tissues, and the expression level of PoCAT mRNA was higher in intestine and mantle. The expression profile analysis showed that the expression level of PoCAT mRNA in intestine was significantly up-regulated at 2, 4 and 12 h after Vibrio alginolyticus stimulation. These results demonstrated that PoCAT is a typical member of catalase family and might be involved in innate immune responses of pearl oyster.

Cloning of a novel glutathione S-transferase 3 (GST3) gene and expressionanalysis in pearl oyster, Pinctada martensii.

Microsomal glutathione S-transferase (MGST) functions in cellular defense against xenobiotics and provides protection against the action of lipid hydroperoxides produced as a consequence of oxidative stress. In this study, a full-length cDNA encoding MGST3 (referred to as PmMGST3) was identified from the pearl oyster, Pinctada martensii by a combination of expressed sequence tag (EST) analysis and rapid amplification of cDNA ends (RACE). The full-length cDNA of PmMGST3 is 971 bp and contains a 5' UTR of 39 bp, a 3' UTR of 491 bp with a canonical polyadenylation signal sequence (AATAAA), and an open reading frame (ORF) of 447 bp encoding a polypeptide of 146 residues. The deduced polypeptide contains a conserved motif (FNCx(1)QRx(2)H) characteristic of the MGST3 subfamily. The PmMGST3 transcript could be detected in all tissues tested, with highest transcript level seen in hepatopancreas. Cadmium treatment significantly increased PmMGST3 mRNA levels in gill and hepatopancreas, while bacterial challenge initially depressed mRNA levels and then increased its level in haemocytes, gill and hepatopancreas in a time-dependent manner. In an assay using cumene hydroperoxide as a substrate, we demonstrated that PmMGST3 possesses glutathione-dependent peroxidase activity. These results suggest that PmMGST3 plays an important role in cellular defense against oxidative stress caused by cadmium and bacteria.

Immunosuppressive effects of environmental stressors on immunological function in Pinctada imbricata.

This study assessed the effects of mechanical agitation, hypo-saline conditions, and exposure to the air on the Akoya pearl oyster, Pinctada imbricata, focusing specifically on the immunological activity of haemocytes. Both phagocytosis and phenoloxidase activity decreased significantly when oysters were exposed to all three stressors. Transient decreases were also evident in total haemocyte counts after mechanical stress and exposure to air, while significant increases in total haemocyte counts were evident after exposure to low salinity. Acid phosphatase activity increased significantly when oysters were exposed to air. The frequency of granulocytes in the haemolymph increased significantly when oysters were stressed by hypo-saline conditions, whilst the relative frequency of granulocytes did not differ significantly after mechanical agitation or exposure to air. The total protein content of haemolymph increased significantly when oysters were stressed by mechanical agitation and low salinity. These results suggest that fluctuations in environmental conditions affect circulating haemocytes and their cytochemistry, and that the different immunological parameters tested were influenced uniquely according to the type of stressor.

Molecular characterization and expression analysis of cathepsin L1 cysteine protease from pearl oyster Pinctada fucata.

Cathepsin L is one of the crucial enzyme superfamilies and involved in the immune responses. In this study, a cDNA encoding cathepsin L cysteine protease was identified and characterized from pearl oyster Pinctada fucata (designated as poCL1). The poCL1 cDNA was 1160 bp long and consisted of a 5'-untranslated region (UTR) of 15 bp, a 3'-UTR of 149 bp with a polyadenylation signal (AATAAA) at 11 nucleotides upstream of the poly(A) tail, and an open reading frame (ORF) of 996 bp encoding a polypeptide of 331 amino acids, which contained a typical signal peptide sequence (Met(1)-Ala(16)), a prodomain (Thr(17)-Asp(113)), and a mature domain (Leu(114)-Val(331)). The preproprotein contained the oxyanion hole (Gln), the active triad formed by Cys, His and Asn, and the conserved ERFNIN, GNFD motifs, which is characteristic for cathepsin L proteases. Homology analysis revealed that the poCL1 shared 62.5-72.5% similarity and 42.9-56.0% identity to other known cathepsin L sequences. The phylogenetic tree showed that the poCL1 clustered with the invertebrate cathepsin L cysteine proteases and was closely related to Stichopus japonicus CL, Strongylocentrotus salar CL1 and Radix peregra CL. The mRNA expression of the poCL1 in blank group and bacterial challenge group could be detected in all studied tissues with the higher level in digestive gland. The expression level of poCL1 mRNA was significantly up-regulated at 4 h and 8 h, and then significantly down-regulated at 12 h and 24 h in digestive gland after Vibrio alginolyticus stimulation. These results provided important information for further exploring the roles of pearl oyster cathepsin L in the immune responses.

Molecular cloning, characterization and expression analysis of a clip-domain serine protease from pearl oyster Pinctada fucata.

The clip-domain serine proteases (SPs) are the essential components of extracellular signaling cascade in various biological processes, especially in embryonic development and the innate immune responses of invertebrate. Herein, we described the isolation and characterization of pearl oyster Pinctada fucata clip-domain SP gene (designated as poSP). The poSP cDNA was 1080 bp long and consisted of a 5'-untranslated region (UTR) of 13 bp, a 3'-UTR of 68 bp with a polyadenylation signal (AATAAA) at 22 nucleotides upstream of the poly(A) tail, and an open reading frame (ORF) of 999 bp encoding a polypeptide of 332 amino acids with an estimated molecular mass of 36.5 kDa and a theoretical isoelectric point of 7.3. A clip-domain and a trypsin-like serine protease domain were identified in the poSP using SMART analysis. Homology analysis of the deduced amino acid sequence of the poSP with other known SP sequences by MatGAT software revealed that the poSP shared 47.0-68.4% similarity to the other known SP sequences. The poSP mRNA was expressed in haemocytes, gonad, digestive gland and mantle, but not expressed in adductor muscle and gill. The poSP mRNA was up-regulated and increased nearly double-fold after LPS or Vibrio alginolyticus stimulation, respectively. These results suggested that the poSP was an inducible acute-phase protein that perhaps involved in the innate immune response of pearl oyster.

Molecular characterization and expression analysis of the IkappaB gene from pearl oyster Pinctada fucata.

Inhibitor of NF-kappaB (IkappaB) is one important member of NF-kappaB signal pathway and plays a pivotal role in regulating the innate immune response of invertebrate. Herein, we described the isolation and characterization of pearl oyster Pinctada fucata IkappaB gene (designated as poIkappaB). The poIkappaB cDNA was 1975 bp long and consisted of a 5' untranslated region (UTR) of 73 bp, a 3' UTR of 807 bp with three RNA instability motifs (ATTTA) and a polyadenylation signal (AATAAA) at 13 nucleotides upstream of the poly (A) tail, and an open reading frame (ORF) of 1095 bp encoding a polypeptide of 364 amino acids with an estimated molecular mass of 40.11 kDa and theoretical isoelectric point of 4.61. A conserved degradation motif (DS(35)GFSS(39)) and six ankyrin repeats were identified in the poIkappaB by SMART analysis. Homology analysis of the deduced amino acid sequence of the poIkappaB with other known IkappaB sequences by MatGAT software revealed that the poIkappaB shared 23.5-63.3% similarities with other known IkappaB isoforms. The poIkappaB mRNA was constitutively expressed in all studied tissues with the most abundant mRNA in the haemocyte. The poIkappaB mRNA was up-regulated and increased 4.13- and 5.28-fold after LPS and Vibrio alginolyticus stimulation, respectively. These results suggested that the poIkappaB was a constitutive and inducible acute-phase protein that perhaps involved in the immune defense of pearl oyster.