Isolation and characterization of homologous TRBP cDNA for RNA interference in Penaeus monodon.

The transactivation response RNA-binding protein (TRBP) interacts with Dicer and binds to double-stranded RNA as a critical component of the RNA-induced silencing complex, which is a key complex in the RNA interference pathway. The full-length cDNA of TRBP from the tiger prawn, Penaeus monodon, (PmTRBP; 1548 bp long with a 1029 bp coding region) was isolated. The encoded polypeptide of 343 amino acids had a predicted molecular mass of 36.8 kDa. Sequence homology and phylogenetic analysis indicated that PmTRBP was evolutionarily closest to TRBP1 from Litopenaeus vannamei, with the three double-stranded RNA-binding motifs that were typical of the TRBP family. Tissue expression profile analysis by quantitative real-time reverse transcription polymerase chain reaction showed that PmTRBP1 was constitutively expressed in all the examined tissues, with a predominant expression in the lymphatic organs and with the weakest expression in the ovaries. Significantly upregulated PmTRBP1 expression was elicited by systemic injections of Staphylococcus aureus, Vibrio vulnificus, and white spot syndrome virus, thereby revealing its pathogen inducibility. Furthermore, exogenous viral nucleoside analogs (high-molecular-weight poly(I:C) dsRNAs as well as R484 single-stranded RNA) were remarkably induced PmTRBP1 transcription at 48 h and 9 h post-injection, respectively, which suggested that PmTRBP1 might function in tiger prawn antibacterial and antiviral response.

Molecular cloning and mRNA expression of M-phase phosphoprotein 6 gene in black tiger shrimp (Penaeus monodon).

It is widely accepted that protein phosphorylation is a major control event in regulating cell cycle. In the present study, a novel M-phase phosphoprotein 6 (MPP6) was identified from black tiger shrimp Penaeus monodon (designated as PmMPP6) by cDNA library and RACE approaches. The full-length cDNA of PmMPP6 was of 690 bp, including a 5'-terminal un-translated region (5'UTR) of 68 bp, a 3'UTR of 172 bp with a poly (A) tail, and an open reading frame (ORF) of 450 bp encoding a polypeptide of 149 amino acids with a predicted molecular weight of 17.01 kDa. Blastx and phylogenetic analysis together supported that PmMPP6 was a novel member of shrimp MPP6. The mRNA expression of PmMPP6 in thirteen tissues was examined by real-time PCR, and mRNA transcript of PmMPP6 was predominantly detectable in tissues of lymphoid and muscle, to a lesser degree in the tissues of gill, ovary and hepatopancreas, and mainly detected in haemocytes, heart and gonad. The temporal expression of PmMPP6 in different developmental stages of ovary was investigated by real-time PCR. During the six stages of ovary development, two peaks expression of PmMPP6 was detected in stage II with 3.78-fold increase and stage V with 3.48-fold increase compared to that in stage I. All these results indicated that PmMPP6 might be involved in regulating shrimp cell cycle and ovary development.

Molecular characterization and expression analysis of lipopolysaccharide and ß-1,3-glucan-binding protein (LGBP) from pearl oyster Pinctada fucata.

The lipopolysaccharide and ß-1,3-glucan-binding protein (LGBP) plays an important function in the innate immune response of invertebrates as a pattern recognition receptor (PRR). Herein, we described the isolation and characterization of pearl oyster Pinctada fucata LGBP (designated as poLGBP). The poLGBP cDNA was 2,075 bp long and consisted of a 5'-untranslated region (UTR) of 18 bp, a 3'-UTR of 299 bp with one cytokine RNA instability motifs (ATTTA), and an open reading frame (ORF) of 1,758 bp encoding a polypeptide of 585 amino acids with an estimated molecular mass of 65.1 kDa and a theoretical isoelectric point of 5.80. Homology analysis of the deduced amino acid sequence of the poLGBP with other known LGBP sequences by MatGAT software revealed that the poLGBP shared 26.3-56.7% identity and 40.5-70.9% similarity to the other known LGBP sequences. SMART and alignment analysis revealed that the poLGBP possessed a potential polysaccharide-binding motif, a glucanase motif, a LPS-binding site, a ß-1,3-linkage of polysaccharide, a glycine-rich region, a threonine-rich region and two N-glycosylation sites. In healthy pearl oyster, the poLGBP mRNA was specifically expressed in digestive gland, and not detected in gill, adductor muscle, gonad, intestine, mantle and hemocytes. However, after bacteria stimulation, the expression of the poLGBP mRNA was significantly up-regulated in digestive gland and also weakly detected in haemocytes, gonad and intestine. After LPS stimulation, the poLGBP mRNA expression was significantly up-regulated at 8 and 12 h in digestive gland, and the expression level was 10.7-fold higher than the PBS group at 12 h. After bacteria stimulation, the expression level of the poLGBP mRNA was also significantly up-regulated in digestive gland and was 12.9-fold higher than the PBS group at 8 h. However, during the experiment, the poLGBP mRNA expression was not detected in gill after LPS or bacteria stimulation. The tissue-specific expression and the expression up-regulation after LPS or bacteria stimulation in digestive gland suggested that the poLGBP was an inducible acute-phase protein and might play an important function in digestion as digestive enzyme and pattern recognition receptor.

Molecular cloning and mRNA expression of cyclophilin A gene in black tiger shrimp (Penaeus monodon).

The techniques of homology cloning and anchored PCR were used to clone the cyclophilin A (CypA) gene from black tiger shrimp (Penaeus monodon). The full-length cDNA of black tiger shrimp CypA (btsCypA) contained a 5' untranslated region (UTR) of 81 bp, an ORF (open reading frame) of 495 bp encoding a polypeptide of 164 amino acids with an estimated molecular mass of 17.68 kDa and a 3' UTR of 308 bp. The predicted amino acid sequence of btsCypA shared high identity with CypA in other organisms. A quantitative reverse transcriptase Real-Time PCR (qRT-PCR) assay was developed to assess the mRNA expression of btsCypA in different tissues and the temporal expression of btsCypA in the hepatopancreas challenged by lipopolyssacharide (LPS). Higher-level mRNA expression of btsCypA was detected in the tissues of hepatopancreas and blood. The expression of btsCypA in the hepatopancreas was up regulated after stimulated by LPS. The results indicated that btsCypA was a constitutive and inducible expressed protein and could be induced by LPS.

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.

Molecular characterization and expression analysis of a putative LPS-induced TNF-alpha factor (LITAF) from pearl oyster Pinctada fucata.

The lipopolysaccharide-induced TNF-alpha factor (LITAF) is a novel transcription factor, which plays an important role in regulating the expression of TNF-alpha and various inflammatory cytokines in response to LPS stimulation and forms a dependent signaling pathway separated from NF-kappaB. Herein, we described the identification and characterization of pearl oyster Pinctada fucata LPS-induced TNF-alpha factor gene (designated as poLITAF). The poLITAF cDNA was 932 bp long and consisted of a 5'-untranslated region (UTR) of 45 bp, a 3'-UTR of 497 bp with two cytokine RNA instability motifs (ATTTA), and an open reading frame (ORF) of 390 bp encoding a polypeptide of 129 amino acids with an estimated molecular mass of 13.5 kDa and a theoretical isoelectric point of 8.36. A LITAF domain at C-terminal was identified in the poLITAF using SMART analysis, which contained two conserved CXXC motifs. Homology analysis of the deduced amino acid sequence of the poLITAF with other known LITAF sequences by MatGAT software revealed that the poLITAF shared 44.2-67.4% similarity and 35.4-50.0% identity to the other known LITAF sequences. The expression level of poLITAF mRNA was the highest in digestive gland and gill, moderate in adductor muscle, gonad, intestine and mantle, the lowest in haemocytes. The poLITAF mRNA expression was significantly up-regulated at 24 h in gill and at 12 h in digestive gland after LPS stimulation respectively. These results suggested that the poLITAF was a constitutive and inducible acute-phase protein that perhaps involved in the innate immune response of 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 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.

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.