Shell repair and the potential microbial causal in a shell disease of the pearl oyster Pinctada fucata.

The pearl oyster Pinctada fucata is famous for producing luxurious pearls. As filter feeders, they are confronted with various infectious microorganisms. Despite a long history of aquaculture, diseases in P. fucata are not well studied, which limits the development of the pearl industry. We report here a shell disease in P. fucata and a study of the shell repair processes. Scanning electron microscopy (SEM) revealed that the nacreous layer gradually recovered from disordered CaCO3 deposition, accompanied by a polymorphic transition from a calcite-aragonite mixture to an aragonite-dominant composition, as revealed by X-ray diffraction analysis. SEM also showed that numerous microbes were embedded in the abnormal shell layers. Similar indications were induced by a high concentration of microbes injected into the extrapallial space, suggesting the potential pathogenic effect of uncontrolled microbes. Furthermore, hemocytes were found to participate in pathogens resistance and might promote shell repair. These results further our understanding of pathogen-host interactions in pearl oysters and have implications for biotic control in pearl aquaculture.

An alpha-2 macroglobulin in the pearl oyster Pinctada fucata: Characterization and function in hemocyte phagocytosis of Vibrio alginolyticus.

Alpha-2 macroglobulin (α2M) is a ubiquitous protease inhibitor and considered to be an evolutionarily conserved constituent of innate host defence system. Here, an α2M gene (designated as Pfα2M) was obtained from the pearl oyster Pinctada fucata by RT-PCR, PCR walking and rapid amplification of cDNA ends (RACE). The Pfα2M cDNA consists of 6394 bp with an open reading frame (ORF) of 5745 bp encoding a protein of 1914 amino acids with a 19 residues signal peptide. Pfα2M sequence contains three putative functional domains, including a bait region, a thiol ester domain and a receptor-binding domain. Phylogenetic analysis revealed that Pfα2M is closely related to the α2Ms from other molluscs. Pfα2M was expressed in all tested tissues including digestive gland, gill, adductor muscle, mantle and foot, while the highest expression was found in hemocytes. Following challenge with Vibrio alginolyticus, Pfα2M expression in hemocytes was significantly up-regulated at 2 h and then returned to the original level at 48 h. Knockdown of Pfα2M by RNA interference significantly reduced the phagocytosis of V. alginolyticus by hemocytes in vivo, and similar results were obtained upon chemical inactivation of the reactive thioester bond in Pfα2M by methylamine treatment. Taken together, it is suggested that Pfα2M is an immune-relevant molecule and involved in phagocytosis of V. alginolyticus by P. fucata hemocytes, and the function of Pfα2M in phagocytosis is dependent on the active thioester bond.

Co-expression of heat shock protein (HSP) 40 and HSP70 in Pinctada martensii response to thermal, low salinity and bacterial challenges.

Heat shock protein (HSP) 40 proteins are a family of molecular chaperones that bind to HSP70 through their J-domain and regulate the function of HSP70 by stimulating its adenosine triphosphatase activity. In the present study, a HSP40 homolog named PmHSP40 was cloned from the hemocytes of pearl oyster Pinctada martensii using EST and rapid amplification of cDNA ends (RACE) techniques. The full-length cDNA of PmHSP40 was 1251 bp in length, which included a 5' untranslated region (UTR) of 75 bp, an open reading frame (ORF) of a 663 bp, and a 3' UTR of 513 bp. The deduced amino acid sequence of PmHSP40 contains a J domain in the N-terminus. In response to thermal and low salinity stress challenges, the expression of PmHSP40 in hemocytes and the gill were inducible in a time-dependent manner. After bacterial challenge, PmHSP40 transcripts in hemocytes increased and peaked at 6 h post injection. In the gill, PmHSP40 expression increased, similar to expression in hemocytes; however, transcript expression of PmHSP40 was significantly up-regulated at 12 h post injection. Furthermore, the transcripts of PmHSP70 showed similar kinetics as that of PmHSP40, with highest induction during thermal, low salinity stress and bacterial challenges. Altogether these results demonstrate that PmHSP40 is an inducible protein under thermal, low salinity and bacterial challenges, suggesting its involvement in both environmental and biological stresses, and in the innate immunity of the pearl oyster.

Transcriptome analysis of the pearl oyster (Pinctada fucata) hemocytes in response to Vibrio alginolyticus infection.

The pearl oyster Pinctada fucata is cultured widely for production of marine pearls in China, while mass mortalities, likely related to pathogenic infections, have occurred frequently in juvenile, mother and operated oysters. To address this issue, understanding host defense mechanisms of P. fucata against pathogenic challenge is extremely important. In the present study, a comparative analysis of hemocyte transcriptomes of P. fucata before and after Vibrio alginolyticus infection was conducted using the Illumina/Hiseq-2000 RNA-Seq technology. A total of 56,345,139 clean reads were generated and then assembled into 74,007 unigenes with an average length of 680 bp and an N50 of 1197 bp. Unigenes were annotated by comparing against non-redundant protein sequence (nr), non-redundant nucleotide (nt), Swiss-Prot, Pfam, Gene Ontology database (GO), Clusters of Orthologous Groups (COG) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases, and 29,615 unigenes (40.01%) were annotated in at least one database. There were 636 genes (518 up-regulated and 118 down-regulated) that were significantly differentially expressed after bacterial challenge, and among which 369 were associated with 122 pathways, including classical immune-related pathways, such as 'MAPK signaling pathway', 'Chemokine signaling pathway', 'Apoptosis' and 'Wnt signaling pathway'. These findings provide information on the pearl oyster innate immunity and may contribute to developing strategies for management of diseases and long-term sustainability of P. fucata culture.

Production and characterization of surfactin-type lipopeptides as bioemulsifiers produced by a Pinctada martensii-derived Bacillus mojavensis B0621A.

Bacillus mojavensis B0621A was isolated from the mantle of a pearl oyster Pinctada martensii collected from South China Sea. Semi-purified surfactins (225 mg L(-1)) were obtained by acid precipitation and vacuum flash chromatography. The component of the semi-purified surfactins was preliminarily analyzed by liquid chromatography mass spectrometer system, and the results showed that all these surfactins could be a group of homologues. Eight surfactin homologues were isolated and afforded by reversed phase high-performance liquid chromatography. Furthermore, their structure was characterized by mass spectrometry analysis combined with nuclear magnetic resonance spectroscopy techniques. These surfactins shared seven amino acids as peptide backbone and a saturated ß-hydroxy fatty acid chain residue (from C13 to C15), differed each other from peptide sequence in the position of Leu7 or Val7. All these surfactins had significant activity and stability of emulsification under various pH (from 7.0 to 12.0), temperature range (from 20 to 115 °C) and sodium chloride concentration (from 2.5 to 20.0 %, w/v). Taken all together, these results indicated that B. mojavensis B0621A have potential to be an alternative source as a biological-derived emulsifying agent.

Production and characterization of Iturinic lipopeptides as antifungal agents and biosurfactants produced by a marine pinctada martensii-derived Bacillus mojavensis B0621A.

Bacillus mojavensis B0621A was isolated from a pearl oyster Pinctada martensii collected from South China Sea. While screening for cyclic lipopeptides potentially useful as lead compounds for biological control against soil-bone fungal plant pathogens, three lipopeptides were isolated and purified from the fermentation broth of B. mojavensis B0621A via vacuum flash chromatography coupled with reversed-phase high performance liquid chromatography (RP-HPLC). The structural characterization and identification of these cyclic lipopeptides were performed by tandem mass spectrometry (MS/MS) combined with gas chromatography-mass spectrometry (GC-MS) analysis as well as chemical degradation. These lipopeptides were finally characterized as homologues of mojavensins, which contained identical amino acids back bones of asparagine1, tyrosine2, asparagine3, glutamine4, proline5, asparagine6, and asparagine7 and differed from each other by their saturated ß-amino fatty acid chain residues, namely, iso-C14 mojavensin, iso-C16 mojavensin, and anteiso-C17 mojavensin, respectively. All lipopeptide isomers, especially iso-C16 mojavensin and anteiso-C17 mojavensin, displayed moderate antagonism and dose-dependent activity against several formae speciales of Fusarium oxysporum and presented surface tension activities. These properties demonstrated that the lipopeptides produced by B. mojavensis B0621A may be useful as biological control agent to fungal plant pathogens.

Gene cloning and function analysis of cytokine-induced suppressor of cytokine signaling (SOCS) from pearl oyster Pinctada fucata.

Cytokine-induced suppressor of cytokine signaling (SOCS) family acts as a negative regulator of cytokine receptor signaling to control excessive cytokine effects and inhibit a variety of signal transduction pathways, particularly the Janus kinases/signal transducers and activators of transcription (JAK/STAT) pathway. In present study, SOCS-2 homolog (PfSOCS-2) from pearl oyster Pinctada fucata was cloned and its gene has no intron. Multiple sequence alignments and phylogenetic analysis showed that PfSOCS-2 was clustered with other mollusk SOCS-2. LPS or polyI:C challenge and gene expression analysis revealed that PfSOCS-2 involved the innate immune response against bacterial and viral infections and that induction of PfSOCS-2 was varied with the different challenge stimulations. Furthermore, Dual-luciferase reporter assays showed that PfSOCS-2 involved in the regulation of vertebrate target genes containing the IFN-stimulated response element or NF-κB binding site in vitro. These results indicated that SOCS-2 from P. fucata plays a regulatory role against the stimulation.

Expression of allograft inflammatory factor-1 (AIF-1) in response to bacterial challenge and tissue injury in the pearl oyster, Pinctada martensii.

Allograft inflammatory factor-1 (AIF-1), an interferon (IFN)-γ-inducible calcium-binding cytokine, is associated with the inflammatory response and defense. We cloned and analyzed the expression pattern of the AIF-1 gene of the pearl oyster Pinctada martensii, hereafter designated PmAIF-1. The full-length PmAIF-1 cDNA is 946 bp in length and consists of a 5'-untranslated region (UTR) of 120 bp, a 3'-UTR of 376 bp, and an open reading frame (ORF) of 450 bp encoding a polypeptide of 149 amino acids with an estimated molecular mass of 17 kDa. Sequence analysis reveals that PmAIF-1 contains two EF hand Ca(+2)-binding motifs like those in previously characterized AIF-1s while alignment with known AIF-1 protein sequences reveals higher similarity to invertebrate orthologs than to those of vertebrates. Quantitative PCR analysis reveals that PmAIF-1 is constitutively expressed, with the highest expression detected in hemocytes, and the expression level of PmAIF-1 mRNA was significantly up-regulated in hemocytes, gill, digestive gland under bacterial challenge and tissue injury. After challenged by gram-negative bacteria Vibrio alginolyticus and Vibrio parahaemolyticus, gram-positive bacteria Bacillus subtilis, the expression level of this gene in hemocytes were all up-regulated and reached the maximum point at 12h (5.80 folds, P<0.01), 6h (5.02 folds, P<0.01) and 12h (5.49 folds, P<0.01), respectively. Under shell damage and mantle injury, PmAIF-1 mRNA increased gradually in the first 3h and reached a peak of expression at 6h post-injury. These findings suggest that PmAIF-1 is an acute-response protein involved in the innate immune responses of pearl oysters, and provide general information about the mechanisms of innate immune defense against bacterial infection in pearl oysters.

Molecular cloning, characterization and expression analysis of tumor necrosis factor receptor-associated factor 3 (TRAF3) from pearl oyster Pinctada fucata.

TRAF3 is a highly versatile regulator that negatively regulates JNK and alternative nuclear factor-κB signalling, but positively controls type I interferon production. To investigate TRAF3 function in innate immune responses among invertebrate especially mollusk, we characterized TRAF3 (PfTRAF3) from pearl oyster Pinctada fucata, one of the most important bivalve mollusks for seawater pearl production. PfTRAF3 cDNA is 2261 bp with an open reading frame of 1623 bp encoding a putative protein of 541 amino acids. The deduced PfTRAF3 contains a RING finger domain, two TRAF domains with zinc finger domains and a conserved C-terminal meprin and TRAF homology (MATH) domain. Comparison and phylogenetic analysis revealed that PfTRAF3 from mollusk shared a higher identity with Ciona intestinalis TRAF3 from urochordata, Branchiostoma belcheri TRAF3 from cephalochordate, and even TRAF3 from vertebrate than with insect homologues. Furthermore, gene expression analyses suggested that PfTRAF3 was involved in the immune response to Vibrio alginolyticus.

Molecular cloning and characterization of class I NF-κB transcription factor from pearl oyster (Pinctada fucata).

NF-κB transcription factors play central roles in many important physiological and pathological processes including innate immune responses. Here we report the cloning of an NF-κB transcription factor, PfRelish from pearl oyster Pinctada fucata, one of the most important bivalve mollusks for seawater pearl production. PfRelish full-length cDNA is 3916 bp with an open reading frame of 3558 bp encoding a putative protein of 1186 amino acids. The deduced PfRelish contains a N-terminal RHD, a nucleus localization signal, an IκB-like domain with six ankyrin repeats and a death domain at the C-terminus, which is similar to class I NF-κB transcription factors. Comparison and phylogenetic analysis revealed that class I NF-κBs in mollusks including PfRelish might have most distant relationship to the arthropod Relish. Further expression analysis showed that PfRelish was apparently upregulated after Vibrio alginolyticus injection, which suggested that PfRelish was involved in the immune response to V. alginolyticus.

Identification of the immune expressed sequence tags of pearl oyster (Pinctada martensii, Dunker 1850) responding to Vibrio alginolyticus challenge by suppression subtractive hybridization.

One hemolymph subtracted cDNA library of pearl oyster (Pinctada martensii, Dunker 1837) was constructed using the suppression subtractive hybridization (SSH) in response to Vibrio alginolyticus. A total of 1089 clones were sequenced. All the consensuses were recognized based on the BLAST searches in NCBI, and revealed that 376 (58%) of them had no significant matches to reported sequences in the database. 267 ESTs were in significant matches after homologous sequence searches. Hypothesized genes inferred from EST sequences were categorized into six groups according to their putative biological functions: replication, transcription and translation; cellular processes; responded to stimuli; metabolism and biosynthesis; signal transduction genes; "other" category. The five genes, pearlin gene promoter PGPPm, serine/threonine kinase STKPm, limbic system-associated membrane protein LSAMPPm, nacrein gene intron 6 NGIPm6 and ferritin-like protein FLPPm, were analyzed using real-time PCR. All these genes were significantly expressed after V. alginolyticus challenge.

A macrophage migration inhibitory factor like oxidoreductase from pearl oyster Pinctada fucata involved in innate immune responses.

Macrophage migration inhibitory factor (MIF) is an important cytokine and plays a crucial role as a pivotal regulator of innate immunity. In this study, a MIF cDNA was identified and characterized from the pearl oyster Pinctada fucata (designated as PoMIF). The full-length of PoMIF was 1544 bp and consisted of a 5'-untranslated region (UTR) of 45 bp, a 3'-UTR of 1139 bp with a polyadenylation signal (AATAAA) at 12 nucleotides upstream of the poly (A) tail. The open reading frame (ORF) of PoMIF was 360 bp which encoded a polypeptide of 120 amino acids with an estimated molecular mass of 13.3 kDa and a predicted pI of 6.1. SMART analysis showed that PoMIF contained the catalytic-sites P² and K³³ for tautomerase activity, a motif C57GSV6° for oxidoreductase activity and a MIF family signature D55PCGSVEVYSIGALG69. Homology analysis revealed that the PoMIF shared 40.3-65.5% similarity and 26.9-45.0% identity to other known MIF sequences. PoMIF mRNA was constitutively expressed in seven selected tissues of healthy pearl oysters, with the highest expression level in digestive gland. Eight hours after P. fucata was injected with Vibrio alginolyticus, the expression of PoMIF mRNA was significantly up-regulated in digestive gland, gills, hemocytes and intestine. The cDNA fragment encoding mature protein of PoMIF was subcloned to expression vector pRSET and transformed into Escherichia coli BL21 (DE3). The recombinant PoMIF (rPoMIF) was expressed and purified under optimized conditions. Function analysis showed that rPoMIF had oxidoreductase activity and could utilize dithiothreitol (DTT) as reductant to reduce insulin.

F-type lectin involved in defense against bacterial infection in the pearl oyster (Pinctada martensii).

In invertebrates and vertebrates, carbohydrate-binding proteins (lectins) play an important role in innate immunity against microbial invasion. In the present study, we report the cloning of an F-type lectin (designated as PmF-lectin) from pearl oyster (Pinctada martensii) using a combination of expression sequence tag (EST) analysis and rapid amplification of cDNA ends (RACE) PCR. The full-length cDNA of PmF-lectin contains an open reading frame (ORF) of 579 bp coding for192 amino acids. The deduced polypeptide possesses six conserved residues of the F-lectin family critical for the formation of disulfide bonds (Cys4³-Cys¹4³, Cys75-Cys76 and Cys¹°²-Cys¹¹9). Reverse transcription PCR (RT-PCR) and real-time quantitative PCR (qRT-PCR) analyses in adult tissues showed that the PmF-lectin mRNA was abundantly expressed in haemocytes and gill, moderately expressed in the mantle, and rarely expressed in other tissues tested. After challenge with Vibrio alginolyticus, expression of PmF-lectin mRNA in haemocytes was dramatically up-regulated, reaching the highest level (13-fold higher than that of the control group) at 3 h post challenge, and then dropped gradually. These results suggest that PmF-lectin is a member of the F-lectin family and is involved in the innate immune response in pearl oyster.

A multidomain galectin involved in innate immune response of pearl oyster Pinctada fucata.

Galectins could specifically bind to ß-galactoside residues and play crucial roles in innate immune responses of vertebrates and invertebrates. In this study, the cDNA of a galectin with multiple carbohydrate-recognition domains (CRDs) was cloned from pearl oyster Pinctada fucata (designated as PoGal). PoGal cDNA was 2138bp long and consisted of a 5'-untranslated region (UTR) of 120bp, a 3'-UTR of 350bp with two cytokine RNA instability motifs (ATTTA), and an open reading frame (ORF) of 1668bp encoding a polypeptide of 555 amino acids with an estimated molecular mass of 63.4kDa and a theoretical isoelectric point of 4.8. PoGal contained four CRDs, each CRD of PoGal all had the conserved carbohydrate-binding motifs H-NPR and WG-ER. PoGal shared 43.7% and 62.9% identity to those of bay scallop and eastern oyster, respectively, which were only two galectins with four CRDs. The phylogenetic analysis revealed that all galectins with four CRDs formed a single clade. PoGal mRNA was constitutively expressed in all detected tissues, and the expression level of PoGal mRNA was significantly up-regulated in digestive gland, mantle, haemocyte, gonad and intestine after Vibrio alginolyticus stimulation. The expression profile analysis showed that the expression level of PoGal mRNA was significantly up-regulated at 4, 8 and 12h after V. alginolyticus stimulation. These results suggested that PoGal was a constitutive and inducible acute-phase protein that perhaps involved in innate immune response of pearl oyster.

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 characterization and expression analysis of interferon-gamma-inducible lysosomal thiol reductase (GILT) gene from pearl oyster Pinctada fucata.

Interferon-gamma-inducible lysosomal thiol reductase (GILT) is an important thiol reductase, involved in class, MHC-restricted antigen processing by catalyzing disulfide bond reduction in mammals. Herein, we describe the identification and characterization of pearl oyster Pinctada fucata GILT (designated as poGILT). The poGILT cDNA was 1273bp long and consisted of a 5'-untranslated region (UTR) of 24bp, a 3'-UTR of 484bp with two cytokine RNA instability motifs (ATTTA), and an open reading frame (ORF) of 765bp encoding a polypeptide of 254 amino acids with an estimated molecular mass of 28.9kDa and a theoretical isoelectric point of 7.4. The N-terminus of the poGILT was found to have a putative signal peptide with a cleavage site amino acid position at 19-20. SMART analysis showed that the poGILT contained a GILT active-site C(69)PDC(72) motif and a GILT signature motif C(115)QHGKEECIGNLIETC(130). Homology analysis of the deduced amino acid sequence of the poGILT with other known GILT sequences by MatGAT software revealed that the poGILT shared 42.9-67.3% similarity and 22.9-49.8% identity to the other known GILT sequences. The expression level of poGILT mRNA was higher in digestive gland, moderate in adductor muscle, gills, gonad, intestine and mantle, and lower in hemocytes. The poGILT mRNA expression was significantly up-regulated in gill and digestive gland after LPS or V. alginolyticus stimulation, respectively. These results suggested that the poGILT was a constitutively expressed acute-phase protein, the expression of which can be enhanced after LPS or V. algrinolyticus stimulation, perhaps involved in the innate immune response of pearl oyster.

Calcineurin mediates the immune response of hemocytes through NF-kappaB signaling pathway in pearl oyster (Pinctada fucata).

Calcineurin (CN), a multifunctional protein, mediates the immune response through diverse signaling pathways in mammals, while the function of CN in the immune response of molluscan hemocytes still remains unclear. In the present study, we detected the distribution of CN in various tissues and the expression levels of Pf-CNA and Pf-CNB gene in hemocytes of Pinctada fucata. After the preparation of hemocyte monolayers, we checked the response of enzymatic activity of CN, the degradation level of IkappaBalpha, the activity of iNOS and the production of NO, and IL-2 to the challenge of lipopolysaccharide (LPS) and cyclosporin A (CsA). CN activity in hemocytes was very sensitive to both the stimulation of LPS and the inhibition of CsA. Most importantly, IkappaBalpha degradation in hemocytes was induced by LPS and attenuated by CsA. Consequently, the activity of iNOS was elevated and the production of NO was increased. Additionally, we found that the synthesis of IL-2 was increased by LPS but was apparently weakened by CsA. In vivo bacterial clearance experiments showed that CsA significantly decreased the ability of in vivo bacteria clearance in pearl oyster. All the results revealed, for the first time, that CN mediated the immune response of molluscan hemocytes via activating NF-kappaB signaling pathway.

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.

Cloning and expression of heat shock protein 70 gene in the haemocytes of pearl oyster (Pinctada fucata, Gould 1850) responding to bacterial challenge.

The cDNA of pearl oyster Pinctada fucata Hsp70 (designated PFHsp70) was cloned by EST and rapid amplification of cDNA ends (RACE) techniques. The full length of PFHsp70 cDNA was 2376 bp, consisting of a 5'-terminal untranslated region (UTR) of 89 bp, a 3' terminal UTR of 328 bp, and an open reading frame (ORF) of 1959 bp encoding a polypeptide of 652 amino acids with a theoretical molecular weight of 71.42 kDa and an estimated isoelectric point of 5.18. BLAST analysis revealed that the PFHsp70 gene shared high similarity with other Hsp70 genes. PFHsp70 contained all the three classical Hsp70 family signatures. The results indicated that the PFHsp70 was a member of the heat shock protein 70 family. Fluorescent real-time quantitative RT-PCR was used to examine the expression of PFHsp70 gene in haemocytes of P. fucata after the challenge of bacteria Vibrio alginolyticus. There was a clear time-dependent expression pattern of PFHsp70 after bacterial challenge, and the mRNA expression reached a maximum level at 4 h post-challenge, which returned to control level after 32 h. The up-regulated mRNA expression of PFHsp70 in P. fucata after bacterial challenge indicates that the Hsp70 gene is inducible and involved in immune response.

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.