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Find video protocols related to scientific articles indexed in Pubmed.
The ferritin gene in ridgetail white prawn Exopalaemon carinicauda: Cloning, expression and function.
Int. J. Biol. Macromol.
PUBLISHED: 09-01-2014
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Ferritin, a primary iron storage protein, plays an important role in iron homeostasis. In this study, a ferritin cDNA (EcFer) with a 516bp open reading frame (ORF) was obtained from Exopalaemon carinicauda (Holthuis) which encodes a 171 amino acid protein. At the 5-terminal untranslated region (5'-UTR), there is a complete iron-responsive element (IRE). EcFer mRNA was mainly expressed in the hepatopancreas and its expression was significantly up-regulated at 12, 24, and 48h after the shrimp was exposed to CuSO4 and CdCl2. The transcript of EcFer was found to be extremely less or even absent at the gastrula and zoea stage. From the egg protozoea stage, the expression of EcFer was significantly up-regulated compared to that of the gastrula stage. In addition, EcFer was successfully expressed in Pichia pastoris and the purified rEcFer by size chromatography could uptake iron. These results suggest that EcFer plays important roles in the iron involved metabolism in shrimp.
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RNA-Seq reveals the dynamic and diverse features of digestive enzymes during early development of Pacific white shrimp Litopenaeus vannamei.
Comp. Biochem. Physiol. Part D Genomics Proteomics
PUBLISHED: 04-17-2014
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The Pacific white shrimp (Litopenaeus vannamei), with high commercial value, has a typical metamorphosis pattern by going through embryo, nauplius, zoea, mysis and postlarvae during early development. Its diets change continually in this period, and a high mortality of larvae also occurs in this period. Since there is a close relationship between diets and digestive enzymes, a comprehensive investigation about the types and expression patterns of all digestive enzyme genes during early development of L. vannamei is of considerable significance for shrimp diets and larvae culture. Using RNA-Seq data, the types and expression characteristics of the digestive enzyme genes were analyzed during five different development stages (embryo, nauplius, zoea, mysis and postlarvae) in L. vannamei. Among the obtained 66,815 unigenes, 296 were annotated as 16 different digestive enzymes including five types of carbohydrase, seven types of peptidase and four types of lipase. Such a diverse suite of enzymes illustrated the capacity of L. vannamei to exploit varied diets to fit their nutritional requirements. The analysis of their dynamic expression patterns during development also indicated the importance of transcriptional regulation to adapt to the diet transition. Our study revealed the diverse and dynamic features of digestive enzymes during early development of L. vannamei. These results would provide support to better understand the physiological changes during diet transition.
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Characterization and function analysis of an anti-lipopolysaccharide factor (ALF) from the Chinese shrimp Fenneropenaeus chinensis.
Dev. Comp. Immunol.
PUBLISHED: 03-19-2014
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Anti-lipopolysaccharide factor (ALF) is one of the widely-studied antimicrobial peptides (AMPs) with broad-spectrum antibacterial activity and antiviral property. Previous studies show the existence of multiform of ALFs in crustacean which are important for immunity of the animals. In the present study, we characterized one isoform of ALF from the Chinese shrimp Fenneropenaeus chinensis (FcALF2). Tissue distribution analysis revealed that FcALF2 showed the highest expression level in the lymphoid organ (Oka) of the shrimp. The expression level of FcALF2 in shrimp was significantly up-regulated when they were injected with Micrococcus lysodeikticus and Vibrio anguillarum. A peptide corresponding to the LPS-binding domain of FcALF2 (FcALF2-LBD) was synthesized to analyze its antimicrobial activities. Data demonstrated that FcALF2-LBD possessed strong antibacterial activity against Gram-positive bacteria Micrococcus luteus and M.lysodeikticus with MIC ranges of 2-4 ?M and 1-2 ?M respectively and significant inhibition activity against white spot syndrome virus (WSSV). The antibacterial activities of the sequence modified peptides (FcALF2-LBDb, FcALF2-LBDv) were apparently enhanced and broadened after the amount of basic amino acids was increased in the synthetic LPS-binding domain. These data provide more insights into understanding the function of LPS-binding domain of ALF and the role of ALF in shrimp immunity.
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Function of shrimp STAT during WSSV infection.
Fish Shellfish Immunol.
PUBLISHED: 03-11-2014
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JAK/STAT signaling pathway plays key roles in the antiviral immunity of mammals, fish and insect. However, limited knowledge is known about the function of JAK/STAT signaling pathway in the antiviral immunity of shrimp although virus disease has caused severe mortality in shrimp aquaculture. In order to understand the function of JAK/STAT signaling pathway in the antiviral immunity of shrimp, dsRNA interfering technique was used to silence the expression of STAT gene in Litopenaeus vannamei, and the mortality of shrimp was detected after WSSV infection. Furthermore, the expressions of some potential target genes regulated by STAT or genes related to RNA interfering pathway were detected in STAT silenced shrimp during WSSV infection. The WSSV copy number in STAT silenced shrimp was 10(2)-10(3) copies/ng DNA which was much lower than that in the control. The mortality in STAT silenced shrimp caused by WSSV infection decreased very significantly compared to their controls. The function of STAT was verified in vitro cultured cells of hematopoietic tissue of crayfish Cherax quadricarinatus by adding specific inhibitor of STAT3(S3I-201), and the cultured cells treated with S3I-201 showed much less WSSV copy number than their controls, which further suggested that STAT might be helpful for the replication of WSSV. Expression analysis on the potential STAT target genes and genes in RNA interfering pathway provide important information for understanding the functional mechanism of STAT in antiviral immunity of shrimp.
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Bioinformatic prediction of WSSV-host protein-protein interaction.
Biomed Res Int
PUBLISHED: 02-05-2014
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WSSV is one of the most dangerous pathogens in shrimp aquaculture. However, the molecular mechanism of how WSSV interacts with shrimp is still not very clear. In the present study, bioinformatic approaches were used to predict interactions between proteins from WSSV and shrimp. The genome data of WSSV (NC_003225.1) and the constructed transcriptome data of F. chinensis were used to screen potentially interacting proteins by searching in protein interaction databases, including STRING, Reactome, and DIP. Forty-four pairs of proteins were suggested to have interactions between WSSV and the shrimp. Gene ontology analysis revealed that 6 pairs of these interacting proteins were classified into "extracellular region" or "receptor complex" GO-terms. KEGG pathway analysis showed that they were involved in the "ECM-receptor interaction pathway." In the 6 pairs of interacting proteins, an envelope protein called "collagen-like protein" (WSSV-CLP) encoded by an early virus gene "wsv001" in WSSV interacted with 6 deduced proteins from the shrimp, including three integrin alpha (ITGA), two integrin beta (ITGB), and one syndecan (SDC). Sequence analysis on WSSV-CLP, ITGA, ITGB, and SDC revealed that they possessed the sequence features for protein-protein interactions. This study might provide new insights into the interaction mechanisms between WSSV and shrimp.
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Granulocytes of the red claw crayfish Cherax quadricarinatus can endocytose beads, E. coli and WSSV, but in different ways.
Dev. Comp. Immunol.
PUBLISHED: 02-03-2014
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The hemocytes of the red claw crayfish Cherax quadricarinatus are classified by morphologic observation into the following types: hyalinocytes (H), semi-granulocytes (SG) and granulocytes (G). Density gradient centrifugation with Percoll was developed to separate these three subpopulations of hemocytes. Beads, Escherichia coli, and FITC labeling WSSV were used to investigate the characteristics of granulocytes by using scanning electron microscope, transmission electron microscope, and laser scan confocal microscope. Results showed that granulocytes could phagocytose beads and E. coli by endocytic pathways. WSSV could rely on caveolae-mediated endocytosis to mainly enter into granulocytes. These results could elucidate the mechanism of the innate immunity function of granulocytes, and it also showed the mechanism by which WSSV invaded granulocytes in the red claw crayfish.
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Modification of a synthetic LPS-binding domain of anti-lipopolysaccharide factor from shrimp reveals strong structure-activity relationship in their antimicrobial characteristics.
Dev. Comp. Immunol.
PUBLISHED: 01-29-2014
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Anti-lipopolysaccharide factor (ALF) is a small protein with broad-spectrum antimicrobial activities and certain antiviral property. Its putative lipopolysaccharide (LPS) binding domain was deduced to be important for its activities. However, there is still no report revealing how the structure of the LPS-binding domain affects its biological function until now. In the present study, we designed and synthesized a peptide corresponding to the LPS-binding domain of ALF from the Chinese shrimp (designated as FcALF-LBDc) and its structure-modified isoforms in order to analyze the relationship between its structure and antimicrobial activities. Results showed that FcALF-LBDc exhibited apparent antibacterial activities against both Gram-negative bacteria Escherichia coli and Vibrio anguillarum and Gram-positive bacteria Micrococcus luteus and Micrococcus lysodeikticus with MIC ranges of 32-64, 2-4, 1-2, and 32-64?M, respectively. The disulfide loop and the basic amino acids in the LPS-binding domain (LBD) of ALF played key roles in its antibacterial activities. In addition, FcALF-LBDc could reduce the propagation of white spot syndrome virus (WSSV) in vivo, and its lysine residue is indispensable for its antiviral property. This is the first attempt to testify the effects of the sequence features of the LPS-binding domain on its antimicrobial activities.
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A new anti-lipopolysaccharide factor (ALF) gene with its SNP polymorphisms related to WSSV-resistance of Litopenaeus vannamei.
Fish Shellfish Immunol.
PUBLISHED: 01-17-2014
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Anti-lipopolysaccharide factors (ALFs) of crustacean play an important role against bacteria or virus infection. In this study, the cDNA sequence and genomic sequence of one new isoform of ALF designated as nLvALF1 were reported. The open reading frame (ORF) of nLvALF1 consisted of 369 bp encoding 123 amino acids and the genomic structure of nLvALF1 comprised four introns and three exons. The predicted pI of the deduced protein was 8.82 and the molecular weight (MW) was 13.72 KDa. The deduced amino acid sequence of nLvALF1 contained a typical functional domain of ALF: LPS-binding domain. Phylogenetic analysis showed that nLvALF1 had the closest relationship with FcALF1 from Fenneropenaeus chinensis. The nLvALF1 was specifically expressed in lymphoid organ (Oka) of shrimp. Its transcriptional level was significantly up-regulated after white spot syndrome virus (WSSV) challenge, suggesting that nLvALF1 might participate in defense against WSSV in Litopenaeus vannamei. In order to search potential genetic markers associated with WSSV-resistance, we scanned the polymorphisms of the genomic fragment with 397 bp where the LPS-binding domain encoding sequence located and 18 SNPs were found. The distribution frequency of these SNPs was analyzed in WSSV susceptible shrimp and resistant shrimp separately. Significant differences existed in allelic frequencies at loci g.1361-T > C, g.1370-T > C, g.1419-T > A between the WSSV-resistant group and the WSSV-susceptible/susceptible group (P < 0.05). The specific haplotype CT consisted of g.1415-C > A and g.1419-T > A was associated with susceptibility to WSSV (P < 0.05). These findings provide theoretical support for selection of WSSV-resistant varieties of L. vannamei.
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A copper-induced metallothionein gene from Exopalaemon carinicauda and its response to heavy metal ions.
Int. J. Biol. Macromol.
PUBLISHED: 01-14-2014
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A full-length copper-induced metallothionein (EcMT-Cu) cDNA was obtained from Exopalaemon carinicauda (Holthuis) and it contained a 198 bp open reading frame that encoded a peptide with 65 amino acid residues. Twenty-one cysteines were found in deduced amino acid sequence and the cysteine (Cys)-rich characteristic was also reported in different types of metallothioneins from other species. EcMT-Cu mRNA expression profile showed that it is the hepatopancreas specific gene. The expression of EcMT-Cu was extremely different when shrimp were exposed to seawater containing 50 ?M CuSO4 or 2.5 ?M CdCl2. The expression of EcMT-Cu in shrimp was significantly up-regulated at 12 and 24 h after exposure to CuSO4, however, its expression was not induced compared to that of pretreatment (p>0.05) when shrimp were exposed to CdCl2. The transcript of EcMT-Cu was found to be extremely low at gastrula and nauplius stage and expression of EcMT-Cu could be detected from egg protozoa stage.
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Molecular characterization, immune response against white spot syndrome virus infection of peroxiredoxin 4 in Fenneropenaeus chinensis and its antioxidant activity.
Fish Shellfish Immunol.
PUBLISHED: 01-10-2014
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Peroxiredoxins (Prx) are a family of antioxidant proteins and perform important functions in intracellular signal transduction. Here, we report a Prx gene from Chinese shrimp Fenneropenaeus chinensis. The full-length cDNA of FcPrx gene contained an open reading frame of 735 bp encoding a polypeptide of 275 amino acids. The molecular mass of the deduced amino acid of FcPrx is 27445.43 Da with an estimated pI of 5.71. Sequence comparison showed that the FcPrx shares high identities with Prx IVs and it was named FcPrx4. A real-Time PCR (qRT-PCR) assay was developed to assess the mRNA expression of FcPrx4 in different tissues and temporal expression in hemocytes and hepatopancreas of F. chinensis challenged by white spot syndrome virus (WSSV). Transcripts of FcPrx4 can be detected in all tissues examined. The expression of FcPrx4 showed significant up-regulation in shrimp hemocytes and hepatopancreas after artificial infection with WSSV. A fusion protein containing FcPrx4 was produced in vitro and was confirmed by Matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) assay. And activity analysis indicated that the recombinant FcPrx4 proteins can reduce H2O2 in the presence of dithiothreitol.
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Acute effects of cadmium and copper on survival, oxygen consumption, ammonia-N excretion, and metal accumulation in juvenile Exopalaemon carinicauda.
Ecotoxicol. Environ. Saf.
PUBLISHED: 01-06-2014
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Ridgetail white prawn (Exopalaemon carinicauda), a commercially important species in China, is a potential candidate for evaluating impairments caused by environmental pollutants in coastal and estuarine areas. The main purpose of the present study was to investigate the acute effects of cadmium (Cd) and copper (Cu) on survival, oxygen consumption, ammonia-N excretion, and metal accumulation in E. carinicauda. The feasibility of using this species for pollution monitoring was also evaluated. Results showed that the median lethal concentrations (LC50) for 24h, 48h, 72h, and 96h were 0.66mg/L, 0.379mg/L, 0.343mg/L, and 0.258mg/L for Cd, and 0.932mg/L, 0.748mg/L, 0.725mg/L, and 0.712mg/L for Cu. Cd exposure (0.66mg/L) caused an inhibition in oxygen consumption of 21.1 percent and an increase in ammonia-N excretion of 47.1 percent, thereby decreasing the atomic ratio of oxygen consumed to nitrogen consumed (O:N ratio) of 46.32 percent relative to the control. Cu exposure (0.932mg/L) also resulted in an inhibition in oxygen consumption of 34.8 percent and a decrease in the O:N ratio of 23.9 percent in relation to the control, but the ammonia-N excretion was not influenced by the Cu exposure. Concentration-depended accumulation was observed in the experimental animals, which a maximum of 244.8 folds and 1.1 folds increase of mental concentration was measured upon exposure to 24h LC50 of Cd and Cu for 24h, respectively. The change in O:N ratio indicated an alteration in energy utilization. Based on its sensitivity to heavy metals and its availability all year round, E. carinicauda can be used as a test organism to monitor for metal pollution.
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Comparative transcriptomic characterization of the early development in Pacific white shrimp Litopenaeus vannamei.
PLoS ONE
PUBLISHED: 01-01-2014
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Penaeid shrimp has a distinctive metamorphosis stage during early development. Although morphological and biochemical studies about this ontogeny have been developed for decades, researches on gene expression level are still scarce. In this study, we have investigated the transcriptomes of five continuous developmental stages in Pacific white shrimp (Litopenaeus vannamei) with high throughput Illumina sequencing technology. The reads were assembled and clustered into 66,815 unigenes, of which 32,398 have putative homologues in nr database, 14,981 have been classified into diverse functional categories by Gene Ontology (GO) annotation and 26,257 have been associated with 255 pathways by KEGG pathway mapping. Meanwhile, the differentially expressed genes (DEGs) between adjacent developmental stages were identified and gene expression patterns were clustered. By GO term enrichment analysis, KEGG pathway enrichment analysis and functional gene profiling, the physiological changes during shrimp metamorphosis could be better understood, especially histogenesis, diet transition, muscle development and exoskeleton reconstruction. In conclusion, this is the first study that characterized the integrated transcriptomic profiles during early development of penaeid shrimp, and these findings will serve as significant references for shrimp developmental biology and aquaculture research.
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Transcriptome analysis of the initial stage of acute WSSV infection caused by temperature change.
PLoS ONE
PUBLISHED: 01-01-2014
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White spot syndrome virus (WSSV) is the most devastating virosis threatening the shrimp culture industry worldwide. Variations of environmental factors in shrimp culture ponds usually lead to the outbreak of white spot syndrome (WSS). In order to know the molecular mechanisms of WSS outbreak induced by temperature variation and the biological changes of the host at the initial stage of WSSV acute infection, RNA-Seq technology was used to analyze the differentially expressed genes (DEGs) in shrimp with a certain amount of WSSV cultured at 18°C and shrimp whose culture temperature were raised to 25°C. To analyze whether the expression changes of the DEGs were due to temperature rising or WSSV proliferation, the expression of selected DEGs was analyzed by real-time PCR with another shrimp group, namely Group T, as control. Group T didn't suffer WSSV infection but was subjected to temperature rising in parallel. At the initial stage of WSSV acute infection, DEGs related to energy production were up-regulated, whereas most DEGs related to cell cycle and positive regulation of cell death and were down-regulated. Triose phosphate isomerase, enolase and alcohol dehydrogenase involved in glycosis were up-regulated, while pyruvate dehydrogenase, citrate synthase and isocitrate dehydrogenase with NAD as the coenzyme involved in TCA pathway were down-regulated. Also genes involved in host DNA replication, including DNA primase, DNA topoisomerase and DNA polymerase showed down-regulated expression. Several interesting genes including crustin genes, acting binding or inhibiting protein genes, a disintegrin and metalloproteinase domain-containing protein 9 (ADAM9) gene and a GRP 78 gene were also analyzed. Understanding the interactions between hosts and WSSV at the initial stage of acute infection will not only help to get a deep insight into the pathogenesis of WSSV but also provide clues for therapies.
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SNP discovery in the transcriptome of white Pacific shrimp Litopenaeus vannamei by next generation sequencing.
PLoS ONE
PUBLISHED: 01-01-2014
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The application of next generation sequencing technology has greatly facilitated high throughput single nucleotide polymorphism (SNP) discovery and genotyping in genetic research. In the present study, SNPs were discovered based on two transcriptomes of Litopenaeus vannamei (L. vannamei) generated from Illumina sequencing platform HiSeq 2000. One transcriptome of L. vannamei was obtained through sequencing on the RNA from larvae at mysis stage and its reference sequence was de novo assembled. The data from another transcriptome were downloaded from NCBI and the reads of the two transcriptomes were mapped separately to the assembled reference by BWA. SNP calling was performed using SAMtools. A total of 58,717 and 36,277 SNPs with high quality were predicted from the two transcriptomes, respectively. SNP calling was also performed using the reads of two transcriptomes together, and a total of 96,040 SNPs with high quality were predicted. Among these 96,040 SNPs, 5,242 and 29,129 were predicted as non-synonymous and synonymous SNPs respectively. Characterization analysis of the predicted SNPs in L. vannamei showed that the estimated SNP frequency was 0.21% (one SNP per 476 bp) and the estimated ratio for transition to transversion was 2.0. Fifty SNPs were randomly selected for validation by Sanger sequencing after PCR amplification and 76% of SNPs were confirmed, which indicated that the SNPs predicted in this study were reliable. These SNPs will be very useful for genetic study in L. vannamei, especially for the high density linkage map construction and genome-wide association studies.
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Structural and functional analysis of the amphioxus IGFBP gene uncovers ancient origin of IGF-independent functions.
Endocrinology
PUBLISHED: 07-11-2013
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IGFs play key roles in regulating vertebrate development, growth, reproduction, and aging. In extracellular fluids, IGFs are bound and regulated by a family of IGF-binding proteins (IGFBPs). Although all known IGFBPs are secreted proteins, some are also found in the nucleus and possess IGF-independent activities. When and how these distinct modes of biological actions have evolved is unknown. In this study, we identified and analyzed an IGFBP gene from amphioxus. Amphioxus shares a common ancestor with the modern vertebrate lineage that dates back to more than 520 million years ago. The amphioxus IGFBP shares all major structural characteristics of vertebrate IGFBPs. Phylogenetic analyses place it in a basal position in the IGFBP lineage. Ligand blot analysis reveals that amphioxus IGFBP does not bind to IGF-I or -II. Changing its Phe70 into Leu, however, is sufficient to convert it into a functional IGF binder. When tested in cultured cells, amphioxus IGFBP is localized in the nucleus, and this is attributed to 2 redundant nuclear localization sequences in its L domain. Furthermore, the amphioxus IGFBP N-terminal domain has strong transcriptional activation activity. Forced expression of amphioxus IGFBP in zebrafish embryos results in dorsalized phenotypes. This action requires nuclear localization. These results suggest that the nuclear localization and transcription activation activity of IGFBPs are ancient functions and the IGF-binding function may have been acquired by opportunistic gain-of-functional mutations later in evolution.
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Comparison of Protein Expression Profiles of the Hepatopancreas in Fenneropenaeus chinensis Challenged with Heat-inactivated Vibrio anguillarum and White Spot Syndrome Virus.
Mar. Biotechnol.
PUBLISHED: 03-23-2013
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Fenneropenaeus chinensis (Chinese shrimp) culture industry, like other Penaeidae culture, has been seriously affected by the shrimp diseases caused by bacteria and virus. To better understand the mechanism of immune response of shrimp to different pathogens, proteome research approach was utilized in this study. Firstly, the soluble hepatopancreas protein samples in adult Chinese shrimp among control, heat-inactivated Vibrio-challenged and white spot syndrome virus-infected groups were separated by 2-DE (pH range, 4-7; sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and pH range, 3-10; tricine-SDS-PAGE). Then the differentially expressed protein spots (?1.5-fold or ?0.67-fold averagely of controls) were analyzed by LC-ESI-MS/MS. Using Mascot online database searching algorithm and SEQUEST searching program, 48 and 49 differentially expressed protein spots were successfully identified in response to Vibrio and white spot syndrome virus infection, respectively. Based on these results, we discussed the mechanism of immune response of the shrimp and shed light on the differences between immune response of shrimp toward Vibrio and white spot syndrome virus. This study also set a basis for further analyses of some key genes in immune response of Chinese shrimp.
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A new shrimp peritrophin-like gene from Exopalaemon carinicauda involved in white spot syndrome virus (WSSV) infection.
Fish Shellfish Immunol.
PUBLISHED: 02-06-2013
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Peritrophin was first separated from insect peritrophic membrane (PM), and it played an important role in stimulating the digestion of food and protecting insects from invasion by microorganisms. In this study, a full-length cDNA of a new peritrophin-like protein (EcPT) was cloned from the ridgetail white shrimp Exopalaemon carinicauda, which was an excellent experimental animal for shrimp. The full length cDNA comprised 1235 bp including an 873 bp open reading frame encoding 291 amino acids. The deduced amino acid sequence contained a segment of signal peptide and three conserved chitin binding type 2 domains (ChtBD2) characterized by having a 6-cysteine motif. Tissue expression analysis revealed that EcPT was mainly expressed in stomach and gills, which were also the two main target tissues of WSSV infection. The transcription levels of EcPT in both stomach and gills were found to have significantly changed upon WSSV infection by real-time PCR. Silencing EcPT by dsRNA interference led to higher survival rate of shrimp against WSSV challenge, which suggested that EcPT might be involved in WSSV infection.
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Transcriptome analysis on Chinese shrimp Fenneropenaeus chinensis during WSSV acute infection.
PLoS ONE
PUBLISHED: 02-05-2013
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Previous studies have discovered a lot of immune-related genes responding to white spot syndrome virus (WSSV) infection in crustacean. However, little information is available in relation to underlying mechanisms of host responses during the WSSV acute infection stage in naturally infected shrimp. In this study, we employed next-generation sequencing and bioinformatic techniques to observe the transcriptome differences of the shrimp between latent infection stage and acute infection stage. A total of 64,188,426 Illumina reads, including 31,685,758 reads from the latent infection group and 32,502,668 reads from the acute infection group, were generated and assembled into 46,676 unigenes (mean length: 676 bp; range: 200-15,094 bp). Approximately 24,000 peptides were predicted and classified based on homology searches, gene ontology, clusters of orthologous groups of proteins, and biological pathway mapping. Among which, 805 differentially expressed genes were identified and categorized into 11 groups based on their possible function. Genes in the Toll and IMD pathways, the Ras-activated endocytosis process, the RNA interference pathway, anti-lipopolysaccharide factors and many other genes, were found to be activated in shrimp from latent infection stage to acute infection stage. The anti-bacterially proPO-activating cascade was firstly uncovered to be probably participated in antiviral process. These genes contain not only members playing function in host defense against WSSV, but also genes utilized by WSSV for its rapid proliferation. In addition, the transcriptome data provides detail information for identifying novel genes in absence of the genome database of shrimp.
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Current status of genetics and genomics of reared penaeid shrimp: information relevant to access and benefit sharing.
Mar. Biotechnol.
PUBLISHED: 01-16-2013
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At present, research and progress in shrimp genomics and genetics show significant developments. Shrimp genetics and genomics also show immense potential for an increased production in a way that meets shrimp culture progress goals for the third millennium. This review article aims to provide an overview of its current status and future direction, discusses questions that need focused research to address them, and summarizes areas where genetics and genomics knowledge can make a positive difference to shrimp culture sustainability. Sustainable progress of penaeid shrimps will depend upon feasible solutions for environmental, research, economic, consumer problems, proper development, and planning policy enforcement. It is recommended that increased funding for biotechnology research and progress be directed to expand worldwide commercial shrimp culture and address environmental and public health issues. For any researcher or shrimp company member who has attempted to or whom would like to thoroughly search the literature to gain a complete understanding of the current state of shrimp genetics and genomics, this publication will be an invaluable source of reference materials, some of which is reported here for the first time.
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Potential relationship among three antioxidant enzymes in eliminating hydrogen peroxide in penaeid shrimp.
Cell Stress Chaperones
PUBLISHED: 11-01-2011
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Antioxidant enzymes, such as glutathione peroxidase (GPx), catalase (CAT), and peroxiredoxin (Prx), are essential components in cells to eliminate excessive reactive oxygen species such as hydrogen peroxide (H(2)O(2)). GPx, CAT, and Prx genes have been reported in penaeid shrimp, and they showed different expression profiles at transcription or protein level when shrimps were challenged by microbes. In order to learn the relationship among the above three genes in their function, GPx, CAT, and Prx transcripts were analyzed, and the variation of GPx and CAT enzyme activity was detected when shrimp was injected with H(2)O(2) or one antioxidant enzyme gene was silenced in shrimp by double-strand RNA injection. The results indicated that there existed some relationships among three antioxidant enzyme genes, CAT, GPx, and Prx in shrimp at transcriptional level. The transcription of CAT and GPx could be directly induced by H(2)O(2) injection, while the transcription of Prx cannot be induced by H(2)O(2). Decreased transcription level of CAT or GPx could lead to increased transcription of the other two genes, which suggested that there existed some compensation among these three antioxidant enzyme genes. These data can help us to understand the roles of antioxidant enzymes in crustacean.
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A BAC-based physical map of Zhikong scallop (Chlamys farreri Jones et Preston).
PLoS ONE
PUBLISHED: 08-28-2011
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Zhikong scallop (Chlamys farreri) is one of the most economically important aquaculture species in China. Physical maps are crucial tools for genome sequencing, gene mapping and cloning, genetic improvement and selective breeding. In this study, we have developed a genome-wide, BAC-based physical map for the species. A total of 81,408 clones from two BAC libraries of the scallop were fingerprinted using an ABI 3130xl Genetic Analyzer and a fingerprinting kit developed in our laboratory. After data processing, 63,641 (?5.8× genome coverage) fingerprints were validated and used in the physical map assembly. A total of 3,696 contigs were assembled for the physical map. Each contig contained an average of 10.0 clones, with an average physical size of 490 kb. The combined total physical size of all contigs was 1.81 Gb, equivalent to approximately 1.5 fold of the scallop haploid genome. A total of 10,587 BAC end sequences (BESs) and 167 markers were integrated into the physical map. We evaluated the physical map by overgo hybridization, BAC-FISH (fluorescence in situ hybridization), contig BAC pool screening and source BAC library screening. The results have provided evidence of the high reliability of the contig physical map. This is the first physical map in mollusc; therefore, it provides an important platform for advanced research of genomics and genetics, and mapping of genes and QTL of economical importance, thus facilitating the genetic improvement and selective breeding of the scallop and other marine molluscs.
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Potential role of cathepsin B in the embryonic and larval development of clam Meretrix meretrix.
J. Exp. Zool. B Mol. Dev. Evol.
PUBLISHED: 01-06-2011
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This study was designed to investigate the possible role of Meretrix meretrix cathepsin B (MmeCB) in embryonic and larval development. MmeCB mRNA expression profile was revealed by semi-quantitative RT-PCR. The level of MmeCB mRNA expression was low in trochophore stage but high in pedveliger stage. MmeCB protein expression was detected in the digestive gland, velum, and epidermis along the edges of the shell in D-larvae and pedveligers by immunocytochemistry. In post larvae, MmeCB protein expression was noticed abundant in the digestive gland, whereas a modest expression was identified in the gill filament. The average shell length of larvae hatched from embryos treated with 0.01, 1, and 10?µmol/L Ca074Me (a cathepsin B inhibitor) was significantly shorter than that of control groups. The metamorphosis rates of larvae treated with 0.01 and 1?µmol/L Ca074Me were significantly lower than that of control groups in 4-day larvae, but not in 5-day larvae. Taken together, these results indicated that MmeCB may have stimulatory effects on embryonic development, metamorphosis, and larval growth during M. meretrix larval development.
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Cloning and expression profiles of two isoforms of a CHH-like gene specifically expressed in male Chinese shrimp, Fenneropenaeus chinensis.
Gen. Comp. Endocrinol.
PUBLISHED: 03-19-2010
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Two full-length cDNA sequences (Fc-CHH1, Fc-CHH2) encoding a crustacean hyperglycemic hormone (CHH) precursor homolog and their DNA sequences were cloned from Chinese shrimp Fenneropenaeus chinensis. The deduced amino acid sequences of them are predicted to contain a signal peptide and a mature peptide. The mature peptides of Fc-CHH1 and Fc-CHH2 shared 78% identity, but they showed low identities (less than 40%) to CHH peptides from other species. Both Fc-CHH1 and Fc-CHH2 proteins contain six highly conserved cysteine residues which are characteristic of the CHH family peptides. The transcripts of Fc-CHH1 and Fc-CHH2 were shown to be specifically present in the spermatophore sac of mature male Chinese shrimp through reverse transcription-polymerase chain reaction (RT-PCR) detection. The transcripts of Fc-CHH1 and Fc-CHH2 begin to appear at the immature stage (115 days after the first post-larvae stage) when the spermatophore sac was first observed to be appeared. In situ hybridization analyses showed that Fc-CHH1 and Fc-CHH2 transcripts located at the epithelial cells in the internal wall of the spermatophore sac. In the cloned DNA sequences of Fc-CHH1 and Fc-CHH2, the predicted transcription factor binding sites in the 5 flanking sequences are different from those previously reported for CHH family genes of crustacean. To our knowledge, these are novel CHH-like genes expressed specifically in male shrimp. Their function needs to be further investigated.
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Proteomic analysis of differentially expressed proteins in lymphoid organ of Fenneropenaeus chinensis response to Vibrio anguillarum stimulation.
Fish Shellfish Immunol.
PUBLISHED: 03-12-2010
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To gain an insight into the function of shrimp lymphoid organ at protein level, we analyzed the proteome of lymphoid organ in healthy Chinese shrimp Fenneropenaeus chinensis (F. chinensis) through two-dimensional gel electrophoresis (2-DE) based proteomic approach. A total of 95 spots representing 75 protein entries were identified by liquid chromatography tandem mass spectrometry (LC-MS/MS) with both online and in-house database. According to Gene Ontology (GO) annotation of biological process, the identified proteins were classified into 13 categories. Among them, approximately 36% of proteins related to cytoskeleton are noticeable. Then, a comparative proteomic approach was employed to investigate the differentially expressed proteins in lymphoid organ of Vibrio anguillarum-challenged F. chinensis. At 24 h post-injection (hpi), 17 differentially expressed protein spots were successfully identified, including 4 up-regulated protein spots (represent 4 proteins: cathepsin L, protein similar to squid CG16901-PC, protein kinase C and protein similar to T-complex Chaperonin 5 CG8439-PA), and 13 down-regulated protein spots (represent 9 proteins: actin, beta-actin, cytoplasmic actin CyII, alpha tubulin, beta tubulin, protein similar to proteasome delta, vacuolar ATP synthase subunit B, elongation factor 2, carboxypeptidase B). These data may help us to understand the function of lymphoid organ and the molecular immune mechanism of shrimp responsive to pathogen infection.
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A Dorsal homolog (FcDorsal) in the Chinese shrimp Fenneropenaeus chinensis is responsive to both bacteria and WSSV challenge.
Dev. Comp. Immunol.
PUBLISHED: 02-16-2010
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Rel/NFkappaB is a family of transcription factors. In the present study, a Rel/NFkappaB family member, Dorsal homolog (FcDorsal) was cloned from the Chinese shrimp Fenneropenaeus chinensis. The full length cDNA of FcDorsal consists of 1627bp, revealed a 1071bp open reading frame encoding 357 aa. The predicted molecular weight (MW) of the deduced amino acid sequence of FcDorsal was 39.78kDa, and its theoretical pI was 8.85. Amino acid sequence analysis showed that FcDorsal contains a Rel homolog domain (RHD) and an IPT/TIG (Ig-like, plexins and transcriptions factors) domain. The signature sequence of dorsal protein existed in the deduced amino acid sequence. Spatial expression profiles showed that FcDorsal had the highest expression level in the hemocytes and lymphoid organ (Oka). The expression profiles in the hemocytes and lymphoid organ were apparently modulated when shrimp were stimulated by bacteria or WSSV. Both Gram-positive (G(+)) bacteria (Micrococcus lysodeikticus) and Gram-negative (G(-)) bacteria (Vibrio anguillarium) injection to shrimp caused the up-regulation of FcDorsal at the transcription level. DsRNA approach was used to study the function of FcDorsal and the data showed that FcDorsal was related to the transcription of Penaeidin 5 in shrimp. The present data provide clues that FcDorsal might play potential important roles in the innate immunity of shrimp. Through comparison of the expression profiles between FcDorsal and another identified Rel/NFkappaB member (FcRelish) in shrimp responsive to WSSV challenge, we speculate that FcDorsal and FcRelish might play different roles in shrimp immunity.
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Physiological and immune responses of Zhikong Scallop Chlamys farreri to the acute viral necrobiotic virus infection.
Fish Shellfish Immunol.
PUBLISHED: 02-12-2010
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The Zhikong Scallop, Chlamys farreri, is one of the most important bivalve mollusks cultured in northern China. However, mass mortality of the cultured C. farreri has posed a serious threat to the maricultural industry in recent years. Acute Viral Necrobiotic Virus (AVNV) is believed as an important etiological agent causing the scallop mass mortalities. To understand the mechanism behind the AVNV associated scallop disease and mortality, we assessed the physiological and immune responses of C. farreri to the virus infection using oxygen consumption rate, ammonium-nitrogen excretion rate, hemocyte copper, zinc superoxide dismutase gene expression, and plasma superoxide dismutase activity and alkaline phosphatase activity as indicators. Scallops challenged by AVNV at 25 degrees C developed typical disease signs 2 days after virus injection. Before the disease manifested, scallop oxygen consumption and NH4+-N excretion rates rose and then fell back. Real-time PCR revealed that the hemocyte cytosol Cu, Zn SOD gene expression was upregulated followed by recovery. The plasma SOD activity, however, augmented consistently following virus injection. Moreover, plasma AKP activity first lowered and then elevated gradually to the highest level at 24 h post virus injection. Scallops challenged by AVNV at 17 degrees C neither developed notable disease nor showed obvious responses that could be associated with the virus infection. While the results suggested a correlation between the elevated seawater temperature and the AVNV infection associated C. farreri mortalities, they also indicated that the viral infection provoked multiple physiological and immune responses in the host scallops.
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Molecular characterization of an ecdysone inducible gene E75 of Chinese shrimp Fenneropenaeus chinensis and elucidation of its role in molting by RNA interference.
Comp. Biochem. Physiol. B, Biochem. Mol. Biol.
PUBLISHED: 02-06-2010
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Ecdysone inducible gene, E75 is a primary target of ecdysone receptor (EcR), and is found to play a critical role in the molting process of arthropods. In this study, a cDNA encoding the E75 of Chinese shrimp Fenneropenaeus chinensis (FcE75) was cloned using RT-PCR and RACE techniques. FcE75 cDNA was 3611bp in length with an ORF of 2394bp. The deduced amino acid sequence of FcE75 had the highest sequence identity to E75 from a land crab Gecarcinus lateralis and E75 of the shrimp Metapenaeus ensis. Quantitative real-time PCR revealed a prominently high expression of FcE75 mRNA in the whole body RNA extract of late premolt period (D3) juvenile shrimp. The role of E75 in the process of shrimp molting was investigated using the RNA interference technique. Long double-stranded RNA corresponding to the FcE75 (dsE75) efficiently silenced the FcE75 transcript levels in juvenile F. chinensis. Further, injection with dsE75 completely arrested the molting process in experimental shrimp which eventually caused death. Setogenic analysis of the uropods from molt-arrested shrimp, showed defective epidermal retraction, poor development of setae and new cuticle. These results indicate that E75 might be related to the molting process and is essential for proper molting and survival of shrimp. This is the first report demonstrating the use of double stranded RNA to elucidate the possible role of E75 in the molting of decapod crustaceans.
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Screening of genes related to ovary development in Chinese shrimp Fenneropenaeus chinensis by suppression subtractive hybridization.
Comp. Biochem. Physiol. Part D Genomics Proteomics
PUBLISHED: 02-01-2010
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The ovary of triploid shrimp Fenneropenaeus chinensis was apparently impaired compared to that of the diploid shrimp at the same age. Therefore triploid shrimp ovary is possible to be taken as a model to understand the mechanism of ovary development of shrimp compared to that of the ovary of diploid shrimp at the same age. In the present study, a suppression subtractive hybridization (SSH) technique was applied to identify differentially expressed genes in the ovary between diploid and triploid shrimp. For the forward library (RNA from the ovary of triploid shrimp as the tester), 54 genes were identified. For the reverse library (RNA from the ovary of diploid shrimp as the tester), 16 genes were identified. The identified genes encoded proteins with multiple functions, including extracellular matrix components, cytoskeleton, cell growth and death, metabolism, genetic information processing, signal transduction/transport or immunity related proteins. Eleven differentially expressed genes were selected to be confirmed in the ovaries of triploid and diploid shrimp by semi-quantitative RT-PCR. Genes encoding spermatogonial stem-cell renewal factor, cytochrome c oxidase subunits I and II, clottable protein, antimicrobial peptide and transposase showed up-regulated expressions in the ovary of triploid shrimp. Genes encoding tubulin, cellular apoptosis susceptibility protein, farnesoic acid O-methyltransferase, thrombospondin and heat shock protein 90 genes showed higher expressions in the ovary of diploid shrimp. The differential expressions of the above genes are suggested to be related to the ovary development of shrimp. It will provide a new clue to uncover the molecular mechanisms underlying the ovarian development in penaeid shrimp.
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Multiple forms of alpha-2 macroglobulin in shrimp Fenneropenaeus chinesis and their transcriptional response to WSSV or Vibrio pathogen infection.
Dev. Comp. Immunol.
PUBLISHED: 01-08-2010
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Alpha-2 macroglobulin (A2M) is a non-specific protease inhibitor involved in host defense. By full length cloning and sequencing we identified three distinct cDNAs for A2M in Chinese shrimp Fenneropenaeus chinesis, designated FcA2M-1, FcA2M-2 and FcA2M-3, respectively. Expression profiles in normal tissues as well as tissues after challenge by white spot syndrome virus (WSSV) and Vibrio pathogen were conducted for FcA2M-1 and FcA2M-2. The FcA2M-1 and FcA2M-2 cDNAs encode proteins with 1501 or 1502 amino acids, respectively, containing the typical conserved domain architecture of A2M. Similar to complement component C3, FcA2M-2 has a catalytic histidine, which may confer opsonic properties on this shrimp A2M. Six variants in the bait region were found in FcA2M-2 responding differently to Vibrio challenge, thereby widening the spectrum of inhibition and the diversity of immune recognition. FcA2M-1 and FcA2M-3, as well as most other protostomia invertebrate A2Ms identified so far, have a serine residue in the catalytic histidine position instead of the conserved asparagine residue found in vertebrate A2Ms. This, as inferred from a carp C3 molecule in which the catalytic histidine is substituted by a serine, suggests A2Ms in lower invertebrates possibly bear C3-like opsonic activity. These FcA2Ms showed much lower similarity to each other than to the A2Ms in other shrimp species, further supported by pylogenetic analysis. FcA2M-1 was found to be expressed most highly in hemocytes and lymphoid organ, while FcA2M-2 was expressed most highly in the heart and lymphoid organ, with the lowest expression in hemocytes. Challenge by WSSV or Vibrio pathogen increased the FcA2M-1 mRNA level in both hemocytes and lymphoid organ. After challenge, FcA2M-2 showed up-regulation in lymphoid organ but not in hemocytes. These expression features indicate that the different types of A2M in F. chinesis carry out different functions and that they are not simply functionally redundant.
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Cloning and expression of glucose regulated protein 78 (GRP78) in Fenneropenaeus chinensis.
Mol. Biol. Rep.
PUBLISHED: 08-21-2009
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GRP78 (78 kDa glucose-regulated protein), also known as BiP (immunoglobulin heavy-chain-binding protein), is an essential regulator of endoplasmic reticulum (ER) homeostasis because of its multiple functions in protein folding, ER calcium binding, and controlling of the activation of transmembrane ER stress sensors. In this report, we cloned the full length cDNA of GRP78 (FcGRP78) from Chinese shrimp Fenneropenaeus chinensis. This cDNA revealed a 2,325 bp with 1,968 bp open reading frame encoding 655 amino acids. This is the first reported GRP78 gene in Crustacea. The deduced amino acid sequence of FcGRP78 shared high identity with previously reported insect GRP78s: 86, 87 and 85% identity with GRP78s of Drosophila melanogaster, Aedes aegypti and Bombyx mori, respectively. Northern blot analysis shows that FcGRP78 is ubiquitously expressed in tissues of shrimp. Heat shock at 35 degrees C significantly enhanced the expression of FcGRP78 at the first hour, reached the maximum at 4 h post heat shock, dropped after that and resumed to the normal level until 48 h of post recovery at 25 degrees C. Additionally, differential expression of FcGRP78 was detected in haemocytes, hepatopancreas and lymphoid organ when shrimp were challenged by white spot syndrome virus (WSSV). We inferred that FcGRP78 may play important roles in chaperoning, protein folding and immune function of shrimp.
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Molecular cloning and characterisation of prophenoloxidase (ProPO) cDNA from Fenneropenaeus chinensis and its transcription injected by Vibrio anguillarum.
Mol. Biol. Rep.
PUBLISHED: 07-25-2009
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The prophenoloxidase(ProPO) gene was cloned from haemocytes of Chinese shrimp Fenneropenaeus chinensis by Rapid Amplification Complementary DNA Ends (RACE) method. The full-length cDNA of prophenoloxidase gene consists of 3040 bp with a 2061 bp Open Reading Frame (ORF), encoding 686 amino acids. Phylogenetic analysis revealed that it belongs to insect-type invertebrate prophenoloxidase gene family. To understand ProPO reaction for pathogenys challenge in shrimp, the expressions of ProPO in different tissues were studied by real-time PCR after challenged by Vibrio anguillarum. The results showed that the expression level of ProPO gene in haemocytes was highest among three studied tissues including haemocytes, lymphoid organ and hepatopancreas. The time-course change of ProPO mRNA levels in challenge experiment showed that ProPO mRNA transcripts had the biggest change extent in lymphoid organ.
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Comparative proteomic profiles of the hepatopancreas in Fenneropenaeus chinensis response to hypoxic stress.
Proteomics
PUBLISHED: 07-07-2009
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Hypoxia, as one suboptimal environmental condition, can affect the physiological state of shrimp during pond aquaculture. To better understand the mechanism of response to hypoxic stress in Chinese shrimp Fenneropenaeus chinensis, proteome research approach was utilized. Differentially expressed proteins of hepatopancreas in adult Chinese shrimp between the control and hypoxia-stressed groups were screened. By 2-DE analysis, 67 spots showed obvious changes after hypoxia. Using LC-ESI-MS/MS, 51 spots representing 33 proteins were identified including preamylase, arginine kinase, phosphopyruvate hydratase, citrate synthase, ATP synthase alpha subunit, chymotrypsin BI, chitinase, ferritin, C-type lectin receptors, transketolase, formylglutathione hydrolase, formyltetrahydrofolate dehydrogenase, aldehyde dehydrogenase, glutathione peroxidase, cytosolic manganese superoxide dismutase, protein disulfide isomerase, beta-actin, oncoprotein nm23, crustacyanin-C1 and so on. These proteins could be functionally classified into several groups such as proteins related to energy production, metabolism-related proteins, immune-related proteins, antioxidant proteins, chaperones, cytoskeleton proteins and ungrouped proteins. The transcription levels of ten selected genes encode the identified proteins were analyzed by real-time PCR at different sampling times of hypoxia. This study is the first analysis of differentially expressed proteins in the hepatopancreas of shrimp after hypoxia and provides a new insight for further study in hypoxic stress response of shrimp at the protein level.
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Molecular cloning and characterisation of a pattern recognition protein, lipopolysaccharide and beta-1,3-glucan binding protein (LGBP) from Chinese shrimp Fenneropenaeus chinensis.
Mol. Biol. Rep.
PUBLISHED: 05-13-2009
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A pattern recognition protein (PRP), lipopolysaccharide and beta-1,3-glucan binding protein (LGBP) cDNA was cloned from the haemocyte of Chinese shrimp Fenneropenaeus chinensis by the techniques of homology cloning and RACE. Analysis of nucleotide sequence revealed that the full-length cDNA of 1,275 bp has an open reading frame of 1,098 bp encoding a protein of 366 amino acids including a 17 amino acid signal peptide. Sequence comparison of the deduced amino acid sequence of F. chinensis LGBP showed a high identity of 94%, 90%, 87%, 72% and 63% with Penaeus monodon BGBP, Litopenaeus stylirostris LGBP, Marsupenaeu japonicus BGBP, Homarus gammarus BGBP and Pacifastacus leniusculus LGBP, respectively. The calculated molecular mass of the mature protein is 39,857 Da with a deduced pI of 4.39. Two putative integrin binding motifs, RGD (Arg-Gly-Asp) and a potential recognition motif for beta-1,3-linkage of polysaccharides were observed in LGBP sequence. RT-PCR analysis showed that LGBP gene expresses in haemocyte and hepatopancreas only, but not in other tissues. Capillary electrophoresis RT-PCR method was used to quantify the variation of mRNA transcription level during artificial infection with heat-killed Vibrio anguillarum and Staphylococcus aureusin. A significant enhancement of LGBP transcription was appeared at 6 h post-injection in response to bacterial infection. These results have provided useful information to understand the function of LGBP in shrimp.
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Identification of a novel relish homolog in Chinese shrimp Fenneropenaeus chinensis and its function in regulating the transcription of antimicrobial peptides.
Dev. Comp. Immunol.
PUBLISHED: 05-01-2009
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Penaeid shrimp, as an invertebrate, relies on the innate immunity to oppose the microbial invaders. Antimicrobial peptides (AMP) are an integral component of the innate immune system in most organisms and function as an early first line of defense against pathogens, but the knowledge about the pathways to regulate the shrimp AMP gene expression is still absent up to date. In the current study, a Relish homolog (FcRelish) was cloned from Chinese shrimp Fenneropenaeus chinensis. The full length cDNA of FcRelish consists of 2157 bp, including 1512 bp open reading frame, encoding 504 amino acids. The predicted molecular weight of FcRelish is 57 kDa, and the theoretical PI is 7.00. Spatial expression profiles showed that FcRelish had the highest expression levels in the hemocytes and lymphoid organ. Both Vibrio anguillarium and Micrococcus lysodeikticus stimulation to shrimp can affect the transcription profile of FcRelish. Silencing of FcRelish through DsRNA interference can greatly change the transcription profile of AMP. Therefore, we suggest that FcRelish identified in the present study is closely related to the transcription of AMP, and then we inferred that Imd pathway might exist in shrimp.
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Identification and characterization of the pathogenic effect of a Vibrio parahaemolyticus-related bacterium isolated from clam Meretrix meretrix with mass mortality.
J. Invertebr. Pathol.
PUBLISHED: 04-22-2009
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A mass mortality of clam, Meretrix meretrix, occurred in Jiangsu Province of China in the late September of 2007. Of the isolates obtained from the diseased clams, MM21 had the strongest virulence to the clam in the virulence test, with a LD50 value of approximately 6 x 10(6)CFU ml(-1). MM21 was identified as Vibrio parahaemolyticus by the VITEK 2 Compact system and 16S rDNA sequencing. Detection of virulence-associated genes by PCR indicated that MM21 was positive for toxR and tlh, and negative for tdh. Compared with control group, histiocytes from MM21-infected clams displayed a variety of cytopathological changes by transmission electron microscopy examination, which included increased lipid droplets in hepatocytes, deposition of granules in the mantle, excessive secretion in the gill. The results of our study suggested that MM21 may have been an etiological element in the mass mortalities of hard clam (M. meretrix) in Jiangsu Province of China in 2007.
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A serpin from Chinese shrimp Fenneropenaeus chinensis is responsive to bacteria and WSSV challenge.
Fish Shellfish Immunol.
PUBLISHED: 04-14-2009
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Arthropod defence responses (e.g. prophenoloxidase (proPO) activation and Toll pathway initiation) are mediated by serine proteinase cascades and regulated by serpins in haemolymph. A serpin (Fc-serpin) cDNA was cloned from the haemocytes of Fenneropenaeus chinensis by rapid amplification of cDNA ends (RACE) PCR and haemocyte cDNA library screening. The full-length cDNA consists of 1734bp, encoding 411 amino acids with a calculated molecular mass of 46.55kDa and a theoretical isoelectric point of 7.70. Fc-serpin contains a typical serpin-like homologue (serine proteinase inhibitors domain). The deduced protein contains a putative signal peptide of 19 amino acids and the serpins signature sequence ((379)FHCNRPFLFLI(389)). Fc-serpin showed some identity with Pacifastacus leniusculus serpin (42%) and Manduca sexta serpin-6 (34%). The reactive centre loop (RCL) sequences of Fc-serpin, P. leniusculus serpin, M. sexta serpin-6 and Bombyx mori serpin-2 are highly similar. An Arg at the P1 position of the reactive site indicates that Fc-serpin may have inhibitory activity against prophenoloxidase activating proteinase (PAP) and clotting enzyme. Transcripts of Fc-serpin mRNA were mainly detected in haemocytes and the lymphoid organ by RT-PCR. The variation of the mRNA transcription level in haemocytes followed by artificial infection with bacteria or white spot syndrome virus (WSSV) was quantified by SYBR Green real-time PCR analysis. Expression profiles of Fc-serpin greatly fluctuated after challenge. This work represents the first report of a serpin in penaeid shrimp. The data provide clues that Fc-serpin might play potential roles in the innate immunity of shrimp.
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Cloning of cytoplasmic heat shock protein 90 (FcHSP90) from Fenneropenaeus chinensis and its expression response to heat shock and hypoxia.
Cell Stress Chaperones
PUBLISHED: 04-11-2009
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Heat shock protein 90 (HSP90) works as a multi-functional chaperone and is involved in the regulation of many essential cellular pathways. In this study, we have identified a full-length complementary DNA (cDNA) of HSP90 (FcHSP90) from Chinese shrimp Fenneropenaeus chinensis. FcHSP90 full-length cDNA comprised 2,552 bp, including a 2,181-bp open reading frame encoding 726 amino acids. Both homology analyses using alignment with previously identified HSP90 and a phylogeny tree indicated that FcHSP90 was a cytoplasmic HSP90. Real-time reverse transcription polymerase chain reaction analysis revealed that FcHSP90 was ubiquitously expressed in all the examined tissues but with highest levels in ovary of F. chinensis. FcHSP90 mRNA levels were sensitively induced by heat shock (from 25 degrees C to 35 degrees C) and reached the maximum at 6 h during heat shock treatment. Under hypoxia conditions, FcHSP90 mRNA levels, in both hemocytes and gill, were induced at 2 h and depressed at 8 h during hypoxia stress. The assessment of FcHSP90 mRNA levels under heat shock and hypoxia stresses indicated that the transcription of FcHSP90 was very sensitive to heat shock and hypoxia, so we deduced that FcHSP90 might play very important roles for shrimp to cope with environmental stress.
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Construction and characterization of a bacterial artificial chromosome (BAC) library of Pacific white shrimp, Litopenaeus vannamei.
Mar. Biotechnol.
PUBLISHED: 02-19-2009
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The pacific white shrimp (Litopenaeus vannamei) is one of the most economically important marine aquaculture species in the world. To facilitate gene cloning and characterization, genome analysis, physical mapping, and molecular selection breeding of marine shrimp, we have developed the techniques to isolate high-quality megabase-sized DNA from hemocyte nuclear DNA of female shrimp and constructed a bacterial artificial chromosome (BAC) genomic library for the species. The library was constructed in the Hind III site of the vector pECBAC1, consisting of 101,760 clones arrayed in 265 384-well microtiter plates, with an average insert size of 101 kb, and covering the genome approximately fivefold. To characterize the library, 92,160 clones were spotted onto high-density nylon filters for hybridization screening. A set of 18 pairs of overgo probes designed from eight cDNA sequences of L. vannamei genes were used in hybridization screening, and 35 positive clones were identified. These results suggest that the shrimp BAC libraries will provide a useful resource for screening of genomic regions of interest candidate genes, gene families, or large-sized synthetic DNA region and promote future works on comparative genomics, physical mapping, and large-scale genome sequencing in the species.
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Identification of a novel inducible cytosolic Hsp70 gene in Chinese shrimp Fenneropenaeus chinensis and comparison of its expression with the cognate Hsc70 under different stresses.
Cell Stress Chaperones
PUBLISHED: 02-18-2009
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The heat shock protein 70 (Hsp70) family is widely expressed in eukaryotic cells as the major chaperone protein. In this study, the full-length complementary DNA (cDNA) of a novel inducible cytosolic Hsp70 family member (FcHsp70) was cloned from Fenneropenaeus chinensis. FcHsp70 full-length cDNA consists of 2,511 bp with a 1,890-bp open reading frame encoding 629 amino acids. Three Hsp70 protein family signatures, IDLGTTYS, IIDLGGGTFDVSIL, and IVLVGGSTRIPKVQK, were found in the predicted FcHsp70 amino acid sequence. Phylogenetic analysis showed that FcHsp70 was categorized together with the inducible HSP70s reported in other crustaceans. Compared to the previously identified cognate Hsp70 (FcHsc70) in F. chinensis, the expression of FcHsp70 showed quite different expression profiles when the shrimp were subjected to different stresses including heat shock and heavy metal treatments. Under heat shock treatment, the expression of FcHsp70 showed much higher up-regulation than FcHsc70. Copper treatment also induced higher up-regulation of FcHsp70 than FcHsc70. Cadmium treatment did not induce the expression of FcHsp70, but caused down-regulation of FcHsc70. The different expression profiles of FcHsp70 and FcHsc70 in shrimp may indicate their different reactions to different stresses. Therefore, Hsp70 or Hsc70 could be developed as a biomarker to indicate different stresses in shrimp.
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Molecular characterization and expression analysis of chitinase (Fcchi-3) from Chinese shrimp, Fenneropenaeus chinensis.
Mol. Biol. Rep.
PUBLISHED: 02-03-2009
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Complementary DNA (cDNA) and genomic DNA, including flanking regions of the chitinase gene (Fcchi-3) of Fenneropenaeus chinensis, were cloned and sequenced. Fcchi-3 was found to have 92.0 and 91.4% identity at the cDNA level to that of Litopenaeus vannamei and Marsupenaeus japonicus, respectively. The predicted amino acid sequence had an overall similarity with a comparable region of L. vannamei (96.8%) and M. japonicus (93.4%). Based on the cDNA sequence, the genomic structure of the gene was characterized. Sequence analysis revealed that the Fcchi-3 gene was composed of seven exons with 411, 252, 186, 132, 171, 117 and 135 bp and six introns with 232, 196, 121, 90, 159 and 157 bp. Analysis by RT-PCR revealed that Fcchi-3 was a hepatopancreas specific gene. Semi-quantitative RT-PCR analysis revealed that Fcchi-3 transcript was down-regulated significantly in response to the challenge of WSSV at 5 h post-injection and then came back to normal level at 37 h. A fusion protein containing Fcchi-3 was produced and the purified recombinant protein exhibited similar biological function. The result of identification through LC-ESI-MS showed that three peptide fragments (-MAADPVLR-, -ATIDPAYNVPELSK- and -AILAVGGWNEGSPK-) of the recombinant protein were identical to the corresponding sequence of L. vannamei chitinase-3. The recombinant Fcchi-3 could degrade the colloid chitin confirming that the recombinant protein is actually the chitinase.
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Molecular characterization and effect of RNA interference of retinoid X receptor (RXR) on E75 and chitinase gene expression in Chinese shrimp Fenneropenaeus chinensis.
Comp. Biochem. Physiol. B, Biochem. Mol. Biol.
PUBLISHED: 01-11-2009
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Retinoid X receptor (RXR)/ultraspiracle (USP) is the heterodimeric partner of ecdysteroid receptor and is required for the molting process of arthropods. To better understand the molecular aspects governing the process of molting in shrimp, the full-length cDNA of two RXRs, named as FcRXR-1 and FcRXR-2 were obtained from Chinese shrimp Fenneropenaeus chinensis which were of 1715 and 1700 bp long, revealed a 1315 and 1300 bp open reading frame (ORF) respectively. Quantitative Real time PCR analysis showed a marked tissue-specific difference in the expression of FcRXR transcript, which revealed that the expression of FcRXR could be regulated in a tissue-specific manner. Moreover, high expression of FcRXR mRNAs was observed in late pre-molt period (D3) and post molt stages (A-B) of shrimp. Among the two isoforms, FcRXR-2 appeared in a considerably high level in all the stages compared to the FcRXR-1. In addition, we examined the temporal expression of two chitinase genes: FcChitinase (FcChi) and FcChitinase-1 (FcChi-1) during the molt cycle of F. chinensis. Both the FcChi and FcChi-1 transcripts were detected in all stages of molting, although considerable fluctuations observed through the molt cycle. Injection of double stranded RXR (dsRXR) into juvenile shrimp resulted in a maximum silencing effect at 48 h post injection. We analyzed the expression levels of FcChi, FcChi-1 and the ecdysone inducible gene E75 (FcE75) in samples of dsRXR injected shrimp. Significant reduction in levels of both FcE75, FcChi and FcChi-1 transcripts occurred in the silenced shrimp. This correlation suggested that RXR might involve in the downstream regulation of E75 and chitinase gene transcription in the ecdysone signaling pathway of decapod crustaceans.
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Cloning, characterization and expression of ferritin subunit from clam Meretrix meretrix in different larval stages.
Comp. Biochem. Physiol. B, Biochem. Mol. Biol.
PUBLISHED: 01-09-2009
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Shell formation is one of the important events during larval development and metamorphosis in bivalves. However, the molecular mechanisms and environmental cues regulating shell initiation and growth are unclear. Here, we report that ferritin, a principal protein for biological iron storage and metabolism, might play a role in larval shell development of the bivalve mollusk Meretrix meretrix. A full-length ferritin subunit cDNA, named as MmeFer, was cloned and characterized. The MmeFer mRNA expression in different developmental stages, from trochophore to post larvae, was analyzed by real-time reverse transcription polymerase chain reaction (RT-PCR). MmeFer mRNA expression in larvae of later developmental stages increased at least 8-fold following trochophores. Moreover, the temporal and spatial expressions of MmeFer mRNA were examined by whole mount in situ hybridization. In the trochophore stage, MmeFer was detectable where it was supposed to be for shell initiation. In the later developmental stages, MmeFer was found near digestive glands and mantle that secret larval shell. MmeFer expression was also detected in larvae cultured in artificial seawater with different iron concentrations ranging from 0 to 100 microM. These results suggest that ferritin may play a role in the shell formation of mollusks.
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cDNA cloning and gene expression pattern following bacterial challenge of peroxinectin in Chinese shrimp Fenneropenaeus chinensis.
Mol. Biol. Rep.
PUBLISHED: 01-07-2009
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Peroxinectin, a cell-adhesive hemoperoxidase that binds superoxide dismutase and mediates blood cells adhesion and migration in invertebrate, is believed to play an important role in cellular immune reaction. In this study, we reported a new peroxinectin gene homologue from Chinese shrimp Fenneropenaeus chinensis. Based on expressed sequence tags (ESTs) of haemocyte cDNA library, we cloned a 2,611 bps full-length cDNA of peroxinectin gene homologue encoded 801 amino acids. Motif scanning of the predicted polypeptide revealed a peroxidase domain and an integrin binding motif (Lys-Gly-Asp, KGD). Peroxinectin gene expressed constitutively in haemocyte as determined by quantitative real-time RT-PCR, the expression level varied following bacterial challenge. These findings suggested that peroxinectin expression is susceptible to exterior stimulus and maintains at a high expression level during bacterial infection.
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An I?B homologue (FcCactus) in Chinese shrimp Fenneropenaeus chinensis.
Dev. Comp. Immunol.
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This study firstly reports the characterization of a functional I?B homologue, FcCactus in Chinese shrimp Fenneropenaeus chinensis. The full length cDNA of FcCactus consists of a 1359 bp open reading frame (ORF) encoding a 453 amino acid protein with a predicted molecular weight (MW) of 48.46 kDa and theoretical pI of 5.23. Phylogenetic analysis and multiple alignments revealed that the deduced amino acid sequence of FcCactus cDNA had high similarities to Cactus or I?B reported in seven other arthropods. Genomic DNA sequence of FcCactus was also obtained with a length of more than 17698 bp and constituted of seven exons and six introns. Analysis on 5-upstream regulatory region of its DNA sequence revealed that it contained the core promoter sequence with the TATA-box and transcription start site existing in it; furthermore, various transcription factor binding sites (HSF, Hb, BR-C Z, Dfd, CF2-II, Croc, Ttk, Dorsal, and c-Rel) were predicted. Spatial expression profiles showed that FcCactus mRNA had the highest expression level in muscle, hemocytes, heart and lymphoid organ. Gram-positive bacteria (Micrococcus lysodeikticus) and Gram-negative bacteria (Vibrio anguillarium) injection to shrimp caused the modulation of FcCactus at the transcription level. DsRNAi (double-strand RNA interference) approach was used to study the function of FcCactus and the data showed that FcCactus could regulate the expression of different antimicrobial peptides (AMPs) and antiviral factor (AV). The present data showed that FcCactus might play important roles in regulating the immune response of shrimp.
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Shrimp MyD88 responsive to bacteria and white spot syndrome virus.
Fish Shellfish Immunol.
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The myeloid differentiation factor 88 (MyD88) is an important adapter protein which links members of the toll-like receptor (TLR) to the downstream components to activate related signaling pathways. In the present study, a MyD88 homolog (FcMyD88) was cloned from penaeid shrimp Fenneropenaeus chinensis. The ORF of FcMyD88 consisted of 1434 bp encoding a polypeptide of 477 amino acids which contains a death domain (DD) and a typical TLR and interleukin-1 receptor (IL-1R)-related (TIR) domain. Homology analysis revealed that the predicted amino acid (aa) sequence of FcMyD88 shared high similarities with a variety of previously reported MyD88s. The time-dependent expression patterns of FcMyD88 in cephalothoraxes of shrimp injected with Vibrio anguillarum (Gram-negative bacteria, G(-)), Micrococcus lysodeikticu (Gram-positive bacteria, G(+)) and white syndrome spot virus (WSSV) were analyzed at transcription and protein level by real-time PCR and western blotting, respectively. The expression level of FcMyD88 mRNA was significantly up-regulated at one hour (h), 12 h and 24 h after stimulation with both V. anguillarum and M. lysodeikticu. The expression level of FcMyD88 protein was 2-fold up-regulated at 12 h post injection (hpi) of inactivated V. anguillarum while it didnt change after M. lysodeikticu injection during this period. After WSSV injection, the expression level of FcMyD88 mRNA remained relatively constant, while the FcMyD88 protein was significantly up-regulated at 12 and 24 hpi. These results suggested that the MyD88-dependent signaling pathway could be involved in the defense of both bacteria and WSSV infection.
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A trehalose-6-phosphate synthase gene from Chinese shrimp, Fenneropenaeus chinensis.
Mol. Biol. Rep.
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Trehalose is an important disaccharide and plays a key role in many organisms under different stress conditions. In the study, a gene (FcTPS) encoded trehalose-6-phosphate synthase was reported from Chinese shrimp, Fenneropenaeus chinensis. The full-length cDNA of FcTPS is 3,281 bp including a poly A-tail of 20 bp, encoding a putative protein of 844 amino acids. The predicted protein contains a glycol_transf_20 domain and a trehalose_PPase domain. Genomic structure of FcTPS is composed of three exons with 192, 157 and 2,912 bp and two introns with 1,057 and 568 bp. In the second intron, four different SSRs are found. Transcripts of FcTPS gene are constitutively expressed in various tissues, with strongest level in hepatopancreas. After the shrimp were challenged with WSSV or Vibrio and the expression of FcTPS in hepatopancreas were analyzed using real-time PCR, the result showed that FcTPS transcript was down-regulated significantly in response to the challenge of Vibro at the early of 5 h post-challenge and then up-regulated significantly at 14 h. In addition, the expression of FcTPS showed the same result after the shrimp were challenged with WSSV. These results provide some new information about the tissue distribution, expression profiles and potential function of the trehalose-6-phosphate synthase in shrimp.
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Functional analysis of the promoter of the heat shock cognate 70 gene of the Pacific white shrimp, Litopenaeus vannamei.
Fish Shellfish Immunol.
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Current knowledge on cis-regulatory elements of immune genes of the Pacific white shrimp (Litopenaeus vannamei) is poor. In this study, we identified the promoter of the heat shock cognate protein 70 (HSC70) gene of L. vannamei (lvhsc70). The promoter activity of lvhsc70 promoter was analyzed in insect sf9 cell lines. First, the putative promoter sequence was proved to be able to drive the expression of reporter EGFP gene successfully. Then serial deletion experiments were conducted to investigate functional transcription elements in the promoter region. The results revealed that both positive and negative transcription elements existed in this region. These results are quite different from the previous report on the promoter of HSC70 gene in Penaeus monodon (pmhsc70), where only positive transcription elements were indicated. The sequences that are not conserved between the promoters of lvhsc70 and pmhsc70 might contribute to the differences. Finally, we tested the effect of a putative "NF-?b binding site" in the promoter and, surprisingly, found that deletion of this site would result in a significantly enhancement of the expression of reporter genes, while the underlying mechanisms remain unrevealed. Our results would provide supports for future studies to identify the functional transcription elements in the lvhsc70 promoter and to expand our knowledge on regulation of innate immune genes in penaeid shrimp.
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Structure and partial protein profiles of the peritrophic membrane (PM) from the gut of the shrimp Litopenaeus vannamei.
Fish Shellfish Immunol.
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Peritrophic membrane (PM) is a non-cellular structure surrounding the food bolus in invertebrates midgut. In this study, the shrimp Litopenaeus vannamei was found continuously secreting a tube-like PM enclosing the fecal pellets. The PM structure was membranous in this penaeid shrimp which was similar to that in Sicyonia ingentis studied and was primarily composed of chitin and proteins. Chitin was detected along the whole PM. By using the approach of gel electrophoresis and mass spectrometry of tryptic peptides, the most extracted proteins from the shrimp PMs were identified mainly including digestion-related, immune-related, antioxidant proteins and proteins related to PM structure. This suggests that PM may participate in modulating its permeability and immobilizating the digestive enzymes, actively protect the gut from pathogen contact, and play an important role in the gut immune system.
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Signaling pathways regulating innate immune responses in shrimp.
Fish Shellfish Immunol.
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The first line of defense against microbial infections in animals is innate immune response which triggers diverse humoral and cellular activities via signal transduction pathways. Toll, IMD and JAK/STAT pathways are regarded as the main pathways regulating the immune response of invertebrates. This paper reviews the main progress of the investigation on the immune response to pathogens infection in shrimp and supposes that these three signal pathways exist in shrimp. Most of the components (proteins or genes) involved in Toll pathway of Drosophila have been cloned also in shrimp which suggested the existence of Toll pathway in shrimp. The data update shows that the Toll pathway of shrimp is responsive not only to Gram-positive bacteria, Gram-negative bacteria, but also to WSSV. Challenge of WSSV can lead to the variation of transcription level of all identified components in shrimp Toll pathway, which supported that Toll pathway in shrimp played important roles during WSSV infection. Two major homologs to the components of IMD pathway of Drosophila, IMD and Relish, have been identified in shrimp, which indicated that IMD pathway should be existed in shrimp and might play important roles in regulating the immune response of shrimp to bacteria and virus infection. Relish in IMD pathway and dorsal in Toll pathway of shrimp were both involved in the immune response of shrimp to bacteria and virus infection, which implied that these two pathways are not completely separated during the immune response of shrimp. The transcription of STAT in shrimp was modulated after WSSV infection, which suggested that a putative JAK/STAT pathway might exist in shrimp and be very important to virus infection. Study on the signaling pathway regulating the immune response in shrimp could help us to understand the innate immune system, and would provide instructions to shrimp disease control. Obviously, to get more clear ideas about the innate immunological pathways in shrimp, more solid functional studies should be done in the future.
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Gene expression profiles of four heat shock proteins in response to different acute stresses in shrimp, Litopenaeus vannamei.
Comp. Biochem. Physiol. C Toxicol. Pharmacol.
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Heat shock proteins (HSPs) are a suite of highly conserved proteins well known for their quick responses to environmental stresses. However, the respective roles of different HSPs in response to a particular environmental stress have not received adequate scientific attentions to date. In this study, the expression profiles of four HSP genes (Lvhsp60, Lvhsp70, Lvhsc70, and Lvhsp90) of the Pacific white shrimp, Litopenaeus vannamei under acute thermal stress, pH challenge, and heavy metal exposure were investigated, respectively, using the quantitative real-time reverse transcription polymerase chain reaction technique. Results showed that the four genes exhibited quite different expression profiles when the shrimp were subjected to each of the above stressors. Under acute thermal stress, the messenger RNA (mRNA) levels of all the four genes were significantly induced, and the transcription level of Lvhsp70 was the most sensitive to temperature fluctuations. Under acute pH challenge, the relative mRNA expression of the four genes was shown to be time and pH dependent, and the strongest response occurred in Lvhsp60. Under acute heavy metal exposure, transcripts of each of the four genes varied depending on metal type and exposure time. Lvhsp60 displayed particularly high sensitivity to cadmium and manganese exposure, while Lvhsp70 showed the most sensitive response to iron and zinc treatments. The results obtained suggest that different LvHSP genes may play different roles in mediating cell stress caused by a specific environmental stressor. Given the response sensitivity and intensity of LvHSP genes to environmental stresses, Lvhsp70 may be most suitable to act as a biomarker indicating thermal stress, iron and zinc stimulation, while Lvhsp60 may be a promising candidate marker of pH stress, cadmium and manganese exposure in shrimp.
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Two spliced variants of insulin-like androgenic gland hormone gene in the Chinese shrimp, Fenneropenaeus chinensis.
Gen. Comp. Endocrinol.
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More and more evidence indicates that the insulin-like androgenic gland hormone (IAG) plays an important role in male sexual differentiation in crustaceans. In the present study, two IAG isoforms (Fc-IAG1 and Fc-IAG2) were identified from the penaeid shrimp Fenneropenaeus chinensis. Sequence analysis of IAG gene (Fc-IAG) showed that Fc-IAG1 and Fc-IAG2 were generated by alternative splicing of Fc-IAG pre-mRNA, and they shared almost the same deduced amino acid sequence. Both of them were composed of signal peptide, B chain, C peptide and A chain. They both contained the six conserved cysteine residues and a putative N-linked glycosylated site like IAGs reported in other crustacean species. Tissue distribution and in situ hybridization analysis revealed that they had the highest expression level in the androgenic gland. The transcripts of Fc-IAG1 and Fc-IAG2 could also be detected in hepatopancreas and nerve cord of both sexes at a low expression level. Analysis on their temporal expression profiles showed that they expressed at all embryonic and post-larvae stages. The expression of Fc-IAG1 at different developmental stages displayed a low and stable manner, while the expression of Fc-IAG2 began to increase from post-larvae stages, which suggested that Fc-IAG2 might be involved in male sexual differentiation. In the 5 flanking sequence of Fc-IAG, putative binding sites for transcription factors regulating transcription of hormone genes and genes related to sexual development were predicted, which provided us a primary understanding on the regulation mechanism of Fc-IAG gene. This is the first time to report the gene structure of IAG gene and distinct variants of IAG transcripts in crustaceans.
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Sequencing and analysis of four BAC clones containing innate immune genes from the Zhikong scallop (Chlamys farreri).
Gene
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The sequencing of BAC clones (~100 kb) can reveal some characteristics of a genome that are challenging to obtain based on short sequences. Additionally, although the immune genes of the Zhikong scallop (Chlamys farreri) have been studied widely, few analyses have been conducted at the DNA level. In this study, four C. farreri BAC clones containing innate immune genes, including hsp70, l gbp (lipopolysaccharide and beta-1,3-glucan binding protein), serine protease and a gene with an immunoglobulin-like domain, were sequenced and analyzed both to explore the genomic characteristics of C. farreri based on long DNA sequences and to promote the study of C. farreri immune genes at the DNA level. The total length of the four BACs was 389.98 kb. A total of 34 genes were predicted in these sequences, and several features of protein-coding regions in the C. farreri genome were inferred based on this information. Two LGBP genes were located close together in a 22-kb region in one BAC clone, indicating the physical linkage of some immune genes in C. farreri. A cluster of membrane transport genes was also observed; these genes might play important roles in eliminating toxins in C. farreri, which lives as a filter feeder. Further analysis showed 15.43% of the BAC sequence was repetitive. Tandem repeats were the most abundant repeat type, followed by transposable elements. A total of 31 SSRs were predicted in the four BACs. An IS10 family transposon was identified, and a suspected regulatory non-coding RNA gene for this transposon (RNA-OUT) was observed to overlap with it complementarily. This work will promote future studies on the genomics, immune system and non-coding regions of C. farreri.
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Three EST-SSR markers associated with QTL for the growth of the clam Meretrix meretrix revealed by selective genotyping.
Mar. Biotechnol.
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The clam Meretrix meretrix is a member of widely cultured, commercially important clams. A marker-trait association analysis was performed using expressed sequence tag (EST) simple sequence repeat (SSR) markers for marker-assisted selection in M. meretrix. Three markers, MM1272, MM2034, and MM7721, were found to be significantly associated with quantitative trait loci (QTLs) controlling shell length (P?
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Recent advances in researches on the innate immunity of shrimp in China.
Dev. Comp. Immunol.
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The annual production of shrimp culture in mainland of China has been over one million tons for several years. The major cultivated penaeidae species are Litopenaeus vannamei, Fenneropenaeus chinensis, Penaeus monodon and Marsupenaeus japonicus. Due to the importance of shrimp aquaculture in China, researchers have paid more attention to the molecular mechanism of shrimp disease occurrence and tried to develop an efficient control strategy for disease. This paper summarizes the research progress related to innate immunity of penaeid shrimp made in the last decade in Mainland China. Several pattern recognition receptors, such as lectin, toll, lipopolysaccharide and ?-1,3-glucan binding protein (LGBP) and tetraspanin were identified. The major signal transduction pathways, including Toll pathway, IMD pathway, which might be involved in the immune response of shrimp, were focused on and most of the components in Toll pathway were identified. Also, cellular immune responses such as phagocytosis and apoptosis were regarded playing very important roles in anti-WSSV infection to shrimp. The molecules involved in the maintenance of the immune homeostasis of shrimp and the progress on molecular structure and pathogenic mechanism of WSSV were summarized. Therefore, the brief outline about the immune system of shrimp is drawn based on the recent data which will help us to understand the immune responses of shrimp to different pathogens.
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