Characterization of Argonaute2 gene from black tiger shrimp (Penaeus monodon) and its responses to immune challenges

Argonaute2 binds to a short guide RNA (microRNA or short interfering RNA) and guides RNAs direct RISC to complementary mRNAs that are targets for RISC-mediated gene silencing. Here we identified and characterized Argonaute2 from black tiger shrimp Penaeus monodon (designated as PmAgo2). The full-length cDNA of PmAgo2 contained a 5′ untranslated region (UTR) of 106 bp, an open reading frame (ORF) of 2616 bp and a 3′ UTR of 123 bp. The predicted PmAgo2 protein is 99.4 KDa with the theoretical isoelectric point of 9.54. PmAgo2 shared the highest similarity of amino acid with Marsupenaeus japonicus Argonaute2 and Litopenaeus vannamei Argonaute2, at 69.0% and 68.5%, respectively. Phylogenic analysis showed PmAgo2 clustered with shrimp Argonaute2, and closed to the group of insects. Real-time quantitative PCR showed that PmAgo2 was widely expressed in almost all examined tissues except eyestalk, with high expression in lymph and haemocyte. mRNA expression also revealed that PmAgo2 was significantly up-regulated by Staphylococcus aureus and White Spot Syndrome Virus (WSSV) in hepatopancreas. Furthermore, our study also confirmed that dsRNA and ssRNA homologous poly (I:C) and R848 activated the expression of PmAgo2. The result indicated that PmAgo2 responded to both bacterial infection and viral infection, especially, it may induce an ssRNA-mediated RNAi with other core members of siRNA pathway in black tiger shrimp.     

Molecular cloning and expression analysis of a heat shock protein (Hsp90) gene from black tiger shrimp (<Emphasis Type="Italic">Penaeus monodon</Emphasis>)

The techniques of homology cloning and anchored PCR were used to clone the Hsp90 gene from black tiger shrimp. The full length cDNA of black tiger shrimp Hsp90 (btsHsp90) contained a 5′ untranslated region (UTR) of 72 bp, an ORF (open reading frame) of 2160 bp encoding a polypeptide of 720 amino acids with an estimated molecular mass of 83-kDa and a 3′ UTR of 288 bp. The sequence of the coding region showed 90 and 84% homology with that of the Chiromantes haematocheir and Homo sapiens, respectively. Conserved signature sequences of Hsp90 gene family were found in the btsHsp90 deduced amino acid sequence. The temporal expressions of Hsp90 gene were constitutively in the black tiger shrimp tissues including liver, ovary, muscle, brain stomach, and heart, and their levels were markedly enhanced after 30-min heat treatment at 37°C. In ovarian maturation stages, the expression of btsHsp90 was strongest in the second stage, weaker in the fourth and first stage.     

White shrimp Litopenaeus vannamei catalase: Gene structure,expression and activity under hypoxia and reoxygenation

Catalase (EC 1.11.1.6) is an antioxidant enzyme involved in redox equilibrium, regulating hydrogen peroxide (H₂O₂) concentration, a harmful reactive oxygen species (ROS) that is produced during hypoxia. Hypoxia occurs commonly in aquatic environments and in shrimp farms. We studied the catalase gene of the shrimp Litopenaeus vannamei and tested its expression and enzyme activity during hypoxia (1.5 mg/L O₂; 6 and 24 h) and reoxygenation (1 h after hypoxia). The complete gene is 2974 bp long and has four introns of 821, 223, 114 and 298 bp, respectively. The first intron has tree microsatellites, with GT and (T)AT(GT) repeated sequences. L. vannamei catalase is part of an invertebrate clade including crustaceans and rotifers. Catalase expression and activity is different in gills and hepatopancreas. Expression in gills increased 3.2 and 3-fold in response to hypoxia and reoxygenation (6 and 24 h hypoxia, followed by 1 h reoxygenation) compared to normoxia, while no differences were detected in the expression and activity in hepatopancreas. Catalase activity in gills had a contrary response to expression in hypoxia and reoxygenation.     

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

Inhibitor of NF-κB (IκB) is one important member of NF-κB 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 IκB gene (designated as poIκB). The poIκB 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 (DS₃₅GFSS₃₉) and six ankyrin repeats were identified in the poIκB by SMART analysis. Homology analysis of the deduced amino acid sequence of the poIκB with other known IκB sequences by MatGAT software revealed that the poIκB shared 23.5–63.3% similarities with other known IκB isoforms. The poIκB mRNA was constitutively expressed in all studied tissues with the most abundant mRNA in the haemocyte. The poIκB mRNA was up-regulated and increased 4.13- and 5.28-fold after LPS and Vibrio alginolyticus stimulation, respectively. These results suggested that the poIκB was a constitutive and inducible acute-phase protein that perhaps involved in the immune defense of pearl oyster.     

Molecular analysis of a ras-like nuclear (Ran) gene from <Emphasis Type="Italic">Penaeus monodon</Emphasis> and its expression at the different ovarian stages of development

In the present study, a ras-like nuclear (Ran) gene was obtained from the ovary and neurosecretory organ in eyestalk cDNA library of black tiger prawn (Penaeus monodon). The full-length black tiger prawn Ran (PmRan) cDNA consisted of 1140 nucleotides including an open reading frame (ORF) 648 bp, a 5′ untranslated region (5′UTR) of 117 bp and a 3′UTR of 375 bp with a polyadenylation signal sequence “aataaa” and a poly (A) tail. The ORF encoded a peptide of 215 amino acids with molecular mass 24.6 kDa and a theoretical isoelectric point of 7.39. ScanProsite analysis indicated that PmRan protein sequence contained a small GTPase Ran family motif. Homology analysis of the deduced amino acid sequence of the PmRan with other known Ran sequences by MatGAT software revealed that the PmRan show very high homology with the sequences of other animals (92.1–98.6% similarity, 85.6–98.1% identity). Analysis of the tissue expression pattern of the PmRan gene showed that the PmRan mRNA was expressed in all tested tissues, including hepatopancreas, ovary, muscle, intestine, neurosecretory organ in eyestalk, neurosecretory organ in brain, stomach, and heart, with the highest levels in ovary. Furthermore, the PmRan expression was found to be high level in the six ovarian stages of development. The results indicated PmRan might play an important role in ovarian development.     

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 12 h (5.80 folds, P < 0.01), 6 h (5.02 folds, P < 0.01) and 12 h (5.49 folds, P < 0.01), respectively. Under shell damage and mantle injury, PmAIF-1 mRNA increased gradually in the first 3 h and reached a peak of expression at 6 h 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 characterization of a Ran isoform gene up-regulated in shrimp immunity

Diseases caused by viruses are the greatest challenge to worldwide shrimp aquaculture. Ran gene was an important antiviral gene identified from shrimp and its mRNA level was up-regulated in response to viral infection. In this investigation, a Ran isoform gene (named Ran-iso) cDNA was cloned from shrimp, Marsupenaeus japonicus. The full-length cDNA of Ran-iso was 1286 bp, including a 5′-terminal untranslated region (UTR) of 272 bp, 3′-terminal UTR of 366 bp and an open reading frame (ORF) of 648 bp encoding a polypeptide of 215 amino acids. The deduced protein was highly homologous, it shared 90.64%, 84.19%, 81.48% and 67.58% identities with Ran protein of shrimp, honey bee, human and tobacco respectively. Ran-iso gene was constitutively expressed in 6 tissues examined, including gill, hepatopancreas, hemolymph, heart, intestine and muscle. However, Ran-iso was highest expressed in hepatopancreas (p < 0.01), whereas the expressions of other five tissues were equal and relatively low. Time course analysis showed that the expression level of Ran-iso was obviously up-regulated 2.8 times (at 6 h) as much as that in the control in the hepatopancreas challenged by WSSV. This investigation might provide a clue to elucidate the shrimp innate immunity and would be helpful to shrimp disease control.     

Identification of reproduction-related proteins and characterization of the protein disulfide isomerase A6 cDNA in ovaries of the giant tiger shrimp Penaeus monodon

Proteomic analysis was carried out for identification of proteins functionally involved in ovarian development of the giant tiger shrimp (Penaeus monodon). A total of 335 protein spots including 183 spots from vitellogenic (stage II) and 152 spots from mature (stage IV) ovaries of intact P. monodon broodstock were examined. Of these, 75 (40.98%) and 59 (38.82%) spots significantly matched known proteins in the databases, respectively. In addition, 270 protein spots including 167 and 103 spots from respective ovarian stages of eyestalk-ablated broodstock were also characterized. A total of 95 (56.89%) and 62 (60.19%) spots matched known proteins, respectively. Among differentially expressed reproduction-related proteins, the full-length cDNA of protein disulfide isomerase A6 (PmPDIA6) was further characterized by RACE-PCR. PmPDIA6 was 1946 bp in length containing an open reading frame (ORF) of 1293 bp corresponding to a polypeptide of 430 amino acids. PmPDIA6 was up-regulated at stage III ovaries in intact shrimp (P < 0.05). Interestingly, eyestalk ablation resulted in a lower expression level of PmPDIA6 in each stage of ovarian development compared to that of intact broodstock (P < 0.05). Results in this study clearly indicated the potential of cellular proteomic studies and gene expression analysis for identification of proteins/genes differentially expressed during ovarian development of P. monodon.     

Molecular cloning of the black tiger shrimp (Penaeus monodon) elongation factor 2 (EF-2): sequence analysis and its expression on the ovarian maturation stage

The techniques of homology cloning and anchored PCR were used to clone the elongation factor 2 (EF-2) gene from black tiger shrimp (Penaeus monodon). The full length cDNA of black tiger shrimp EF-2 (btsEF-2) contained a 5' untranslated region (UTR) of 73 bp, an ORF of 2541 bp encoding a polypeptide of 846 amino acids with an estimated molecular mass of 95 kDa, and a 3( UTR of 112 bp. The searches for protein sequence similarities with BLAST analysis indicated that the deduced amino acid sequence of btsEF-2 was homological to the EF-2 of other species and even the mammalians. The conserved signature sequence of EF-2 gene family, GTPase effector domain and ADP-ribosylation domain were found in the btsEF-2 deduced amino acid sequence. The temporal expressions of gene in the different ovarian stages were measured by real time PCR. The mRNA expressions of the gene were constitutively expressed in ovary and different during the maturation stages. The result indicated that EF-2 gene was constitutively expressed and could play a critical role in the ovarian maturation stage.