Cloning and expression of glucose regulated protein 78 (GRP78) in Fenneropenaeus chinensis

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 35A 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 25A 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.

Molecular cloning and stress-dependent expression of a gene encoding Delta(12)-fatty acid desaturase in the Antarctic microalga Chlorella vulgaris NJ-7

The psychrotrophic Antarctic alga, Chlorella vulgaris NJ-7, grows under an extreme environment of low temperature and high salinity. In an effort to better understand the correlation between fatty acid metabolism and acclimation to Antarctic environment, we analyzed its fatty acid compositions. An extremely high amount of Delta(12) unsaturated fatty acids was identified which prompted us to speculate about the involvement of Delta(12) fatty acid desaturase in the process of acclimation. A full-length cDNA sequence, designated CvFAD2, was isolated from C. vulgaris NJ-7 via reverse transcription polymerase chain reaction (RT-PCR) and RACE methods. Sequence alignment and phylogenetic analysis showed that the gene was homologous to known microsomal Delta(12)-FADs with the conserved histidine motifs. Heterologous expression in yeast was used to confirm the regioselectivity and the function of CvFAD2. Linoleic acid (18:2), normally not present in wild-type yeast cells, was detected in transformants of CvFAD2. The induction of CvFAD2 at an mRNA level under cold stress and high salinity is detected by real-time PCR. The results showed that both temperature and salinity motivated the upregulation of CvFAD2 expression. The accumulation of CvFAD2 increased 2.2-fold at 15A degrees C and 3.9-fold at 4A degrees C compared to the alga at 25A degrees C. Meanwhile a 1.7- and 8.5-fold increase at 3 and 6% NaCl was detected. These data suggest that CvFAD2 is the enzyme responsible for the Delta(12) fatty acids desaturation involved in the adaption to cold and high salinity for Antarctic C. vugaris NJ-7.

Molecular characterization and effect of RNA interference of retinoid X receptor (RXR) on E75 and chitinase gene expression in Chinese shrimp Fenneropenaeus chinensis

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. (C) 2009 Published by Elsevier Inc.

Identification and expression of TRAF6 (TNF receptor-associated factor 6) gene in Zhikong Scallop Chlamys farreri

Tumor necrosis factor receptor-associated factor 6 (TRAF6), a key signaling adaptor molecule common to the TNFR superfamily and IL-IR/TLR family, is important not only for a diverse array of physiological processes functions of the TNFR superfamily, but also is involved in adaptive immunity and innate immunity. In this report, the first bivalve TRAF6 (named as CfTRAF6) gene is identified and characterized from Zhikong scallop Chlamys farreri. The full-length cDNA of CfTRAF6 is of 2510 bp, consisting of a 5'-terminal untranslated region (UTR) of 337 bp, a 3'-terminal UTR of 208 bp with a canonical polyadenylation signal sequence AATAAA and a poly (A) tail, and an open reading frame (ORF) encoding a polypeptide of 655 amino acids. The predicted amino acid sequence of CfTRAF6 comprises characteristic motifs of the TRAF proteins, including a Zinc finger of RING-type, two Zinc fingers of TRAF-type, a coiled-coil region, and a MATH (the meprin and TRAF homology) domain. The overall amino acid sequence identity between CfTRAF6 and other TRAF6s is 28-68%. Phylogenetic analyses of CfTRAF6 sequence with TRAF sequences from other organisms indicate that CfTRAF6 is a true TRAF6 orthologue. The mRNA expression of CfTRAF6 in various tissues is measured by Real-time RT-PCR. The mRNA transcripts are constitutively expressed in tissues of haemocyte, muscle, mantle, heart, gonad and gill, but the highest expression is observed in the gonad. The temporal expressions of CfTRAF6 mRNA in the mixed primary cultured haemocytes are recorded after treatment with 20 mu g mL(-1) and 0.5 mu g mL(-1) peptido-glycan (PGN). The expression level of CfTRAF mRNA is down-regulated from 1.5 h to 3 h after the treatment with 0.5 mu g mL(-1) PGN, and then recovers to the original level. While the expression of CfTRAF6 is obviously decreased after treatment with 20 mu g mL(-1) PGN, and reach the lowest point (only about 1/9 times to control) at 3 h. The result Suggests that CfTRAF6 can be greatly regulated by PGN and it may be involved in signal transduction and immune response of scallop. (C) 2008 Published by Elsevier Ltd.

Identification of an Atlantic salmon IFN multigene cluster encoding three IFN subtypes with very different expression properties

A cluster of 11 interferon (IFN) genes were identified in the Atlantic salmon genome linked to the growth hormone I gene. The genes encode three different IFN subtypes; IFNa (two genes), IFNb (four genes) and IFNc (five genes), which show 22-32% amino acid sequence identity. Expression of the fish IFNs were studied in head kidney, leukocytes or To cells after stimulation with the dsRNA poly I:C or the imidazoquinoline S-27609. In mammals, poly I:C induces IFN-beta through the RIG-I/MDA5 or the TLR3 pathway, both of which are dependent on NF-kappa B. In contrast, S-27609 induces mammalian IFN-alpha in plasmacytoid dendritic cells through the TLR7 pathway independent of NF-kappa B. The presence of an NF-kappa B site in their promoters and their strong up-regulation by poly I:C, suggest that salmon IFNa1/IFNa2 are induced through similar pathways as IFN-beta. In contrast, the apparent lack of NF-kappa B motif in the promoter and the strong upregulation by S-27609 in head kidney and leukocytes, suggest that IFNb genes are induced through a pathway similar to mammalian IFN-alpha. IFNc genes showed expression patterns different from both IFNa and IFNb. Taken together, salmon IFNa and IFNb are not orthologs of mammalian IFN-beta and IFN-alpha, respectively, but appear to utilize similar induction pathways. (C) 2008 Elsevier Ltd. All rights reserved.

Cloning, characterization and expression of ferritin subunit from clam Meretrix meretrix in different larval stages

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 mu M. These results suggest that ferritin may play a role in the shell formation of mollusks. (C) 2009 Elsevier Inc. All rights reserved.

Expression pattern of dmrt4 from olive flounder (Paralichthys olivaceus) in adult gonads and during embryogenesis

The dmrt (doublesex and mab-3 related transcription factor) gene family comprises several transcription factors that share a conserved DM domain. Dmrt1 is considered to be involved in sexual development, but the precise function of other family members is unclear. In this study, we isolated genomic DNA and cDNA sequences of dmrt4, a member of the dmrt gene family, from olive flounder, Paralichthys olivaceus, through genome walking and real-time reverse transcriptase (RT)-PCR. Sequence analysis indicated that its genomic DNA contains two exons and one intron. A transcriptional factor binding sites prediction program identified a sexual development-related protein, Sox9 (Sry-like HMG box containing 9) in its 5' promoter. Protein alignment and phylogenetic analysis suggested that flounder Dmrt4 is closely related to tilapia Dmo (DM domain gene in ovary). The expression of dmrt4 in adult flounder was sexually dimorphic, as shown by real-time RT-PCR analysis, with strong expression in the testis but very weak expression in the ovary. Its expression was also strong in the brain and gill, but there was only weak or no expression at all in some of the other tissues tested of both sexes. During embryogenesis, its expression was detected in most developmental stages, although the level of expression was distinctive of the various stages. Whole mount in situ hybridization revealed that the dmrt4 was expressed in the otic placodes, forebrain, telencephalon and olfactory placodes of embryos at different developmental stages. These results will improve our understanding of the possible role of flounder dmrt4 in the development of the gonads, nervous system and sense organs.

Suppression of zebrafish VEGF gene by cytomegalovirus promoter-driven short hairpin constructs induces vascular development defects and down regulation NRP1 expression

The usage of RNA interference for gene knockdown in zebrafish through expression of the small interfering RNA mediators from DNA vectors has created a lot of excitement in the research community. In this work, the ability of human cytomegalovirus immediate early promoter (CMV promoter)-driven short hairpin RNA (shRNA) expression vector to induce shRNA against vascular endothelial growth factor (VEGF) gene in zebrafish was tested, and its effects on VEGF-mediated vasculogenesis and angiogenesis were evaluated. Altogether four vectors targeting various locations of VEGF gene were constructed, and pSI-V4 was proven to be the most effective one. Microinjection of pSI-V4 into the zebrafish embryos resulted in defective vascular formation and down regulation of VEGF expression. In situ hybridization analysis indicated that silencing VEGF gene expression by pSI-V4 resulted in down regulation of neuropilin-1 (NRP1), a potent VEGF receptor. Knockdown of VEGF expression by morpholino gave the same result. This provided evidence that the VEGF-mediated angiogenesis in zebrafish was in part dependent on NRP1 expression. The results contributed to a better understanding of molecular mechanisms of cardiovascular development and provided a potential promoter for making inducible knockdown in zebrafish.

VEGF gene silencing by cytomegalovirus promoter driven ShRNA expression vector results in vascular development defects in zebrafish

Zebrafish has been generally considered as an excellent model in case of drug screening, disease model establishment, and vertebrate embryonic development study. In this work, the ability of human cytomegalovirus immediate early promoter (CMV promoter)-driven short hairpin RNA (shRNA) expression vector to induce shRNA against VEGF gene in zebrafish was tested, and its effect on vascular development was assed, too. Using RT-qPCR, blood vessel staining, and in situ hybridization, we confirmed certain transcriptional activity and down regulation of gene expression by the vector. In situ hybridization analysis indicated selective inhibition of NRP1 expression in the VEGF gene loss of function model, which might imply in turn that VEGF could not only activate endothelial cells directly but also could contribute to stimulating angiogenesis in vivo by a mechanism that involved up-regulation of its cognate receptor expression in zebrafish. This contributed to a better understanding of molecular mechanisms of cardiovascular development. The system improved the success rate in making inducible knockdown and widened the possibilities for better therapeutic targets in zebrafish.

Arginine kinase from Litopenaeus vannamei: Cloning, expression and catalytic properties

Arginine kinase (AK) is a phosphotransferase that plays a critical role in energy metabolism in invertebrates. in this paper, the full-length cDNA of AI( was cloned from shrimp, Litopenaeus vannamei by using RT-PCR and RACE PCR. It was 1446 bp encoding 356 amino acids, and belongs to the conserved phosphagen kinase family. The quantitative real-time reverse transcription PCR analysis revealed a broad expression of AK with the highest expression in the muscle and the lowest in the skin. The expression of AK after challenge with LIPS was tested in hemocytes and muscle, which indicated that the two peak values were 6.2 times (at 3 h) and 10.14 times (at 24 h) in the hemocytes compared with the control values, respectively (P < 0.05), while the highest expression of AK was 41 times (at 24 h) in the muscle compared with the control (P < 0.05). In addition, AK was expressed in Eschetichia coli by prokaryotic expression plasmid pGEX-4T-2. The recombinant protein was expressed as glutathione s-transferase (GST) arginine kinase (GST-AK) fusion protein, which was purified by affinity chromatography using Glutathione Sepharose 4B. After cleavage from GST by using a site-specific protease, the recombinant protein was identified by ESI-MS and showed AK activity. After treatment with 10 mM ATP, the enzyme activity significantly increased. However, the enzyme activity was inhibited by 10 mM alpha-ketoglutarate, 50 mM glucose and 200 mM ATP. This research suggested that AK might play an important role in the coupling of energy production and utilization and the immune response in shrimps. (C) 2009 Elsevier Ltd. All rights reserved.

Expression profiles of penaeidin from Fenneropenaeus chinensis in response to WSSV and vibrio infection by real-time PCR

Penaeidin from Chinese shrimp (Fenneropenaeus chinensis) has proved to be one of the most important antimicrobial peptides in the bodies of animals. The relative quantitative real-time PCR method is developed to study through time, the mRNA expression profile of penaeidin in the muscle and haemocyte tissue of Chinese shrimp infected with vibrio (Vibrio anguillarum) and WSSV (white spot syndrome virus). Research results showed that the same pathogens infection experiments produced similar gene expression profile in different tissues while different expression profiles appeared in the same tissues infected by different exterior pathogens. In vibrio infection experiments, a "U" Re expression profile resulted. Expression levels of penaeidin increased and surpassed the non-stimulated level, indicating that penaeidin from Chinese shrimp has noticeable antimicrobial activities. In WSSV infection experiments, the expression profile appeared as an inverse "U" with the expression of penaeidin gradually decreasing to below baseline level after 24 h. The expression of antimicrobial peptides gene in mRNA level in response to virus infection in shrimp showed that international mechanisms of virus to haemocytes and microbial to haemocytes are completely different. Decline of penaeidins expression levels may be due to haemocytes being destroyed by WSSV or that the virus can inhibit the expression of penaeidins by yet undiscovered modes. The expression profiles of penaeidin in response to exterior pathogen and the difference of expression profiles between vibrio and WSSV infection provided some clues to further understanding the complex innate immune mechanism in shrimp.

High level expression, purification, and characterization of the shrimp antimicrobial peptide, Ch-penaeidin, in Pichia pastoris

Penaeidins, members of a new family of antimicrobial peptides constitutively produced and stored in the haemocytes of penaeid shrimp, display antimicrobial activity against bacteria, and fungi. Here, a DNA sequence encoding the mature Ch-penaeidin peptide was cloned into the pPIC9K vector and transformed into Pichia pastoris. The transformed cells were screened for multi-copy plasmids using increasing concentrations of G418. Positive colonies carrying chromosomal integrations of the Chp gene were identified by phenotype and PCR. When transformed cells were induced with methanol, SDS-PAGE and Western blotting revealed the production of a similar to6100 Da recombinant CHP (rCHP) expression product. Large scale expression revealed that rCHP was produced at 108 mg/L under optimal conditions in the highest Chp-producing P. pastoris clone. The antimicrobial activities of rCHP were studied by liquid phase analysis, which revealed that rCHP exhibited activities against some Gram-negative and Gram-positive bacteria, but had a relatively low activity against some fungi. Purification of rCHP by cation exchange chromatography and subsequent automated amino acid sequencing revealed the presence of four additional amino acids (YVEF) at the N-terminus that belonged to the cleaved fusion signal peptide; these residues may account for the observed decrease in antifungal activity. Together, these observations indicate that rCHP is an effective antimicrobial peptide that can be successfully produced at high levels in the yeast, and therefore may be a potential antimicrobial candidate for practical use. (C) 2004 Elsevier Inc. All rights reserved.

Molecular cloning and expression profile of putative antilipopolysaccharide factor in Chinese shrimp (Fenneropenaeus chinensis)

A new antimicrobial protein gene of the anti-lipopolysaccharide factor family (tentatively named as ALFFc) has been cloned from hemocytes of the Chinese shrimp Fenneropenaeus chinensis by rapid amplification of 3' and 5' complementary DNA ends with polymerase chain reaction. The full-length complementary DNA of ALFFc consists of 600 bp with a 369-bp open reading frame, encoding 123 amino acids. The deduced peptide contains a putative signal peptide of 25 amino acids and mature peptide of 98 amino acids. The molecular mass of the deduced mature peptide is 13799.16 Da. It is highly cationic, with a theoretical pI of 10.3. The deduced amino acid sequence of ALFFc showed 56% homology with sequences of Tachypleus tridentatus and L. polyhemus. The tissue expression profile of this gene was studied by Northern blot, and ALFFc transcripts were mainly detected in hemocytes, gill, and intestine. RNA in situ hybridization showed that ALFFc was constitutively expressed in hemocytes. Capillary electrophoresis reverse transcriptase PCR was used to quantify the variation of messenger RNA transcription level during the artificial infection process with Vibrio anguillarum. Significant enhancement of ALFFc transcription appeared during the first 24 hours in response to Vibrio infection. These results provide useful information for understanding the function of ALFFc in shrimp.

cDNA cloning and mRNA expression of the translationally controlled tumor protein (TCTP) gene from Japanese sea perch (Lateolabrax japonicus)

A homologue of the lower vertebrates translationally controlled tumor protein (TCTP) was cloned from the marine fish Japanese sea perch (Lateolabrax japonicus) by the technology of homology cloning. The full-length cDNA sequence of the sea perch TCTP gene contained a 5' untranslated region (UTR) of 47 bp, a 3' UTR of 433 bp, and a putative open reading frame (ORF) of 510 bp encoding a polypeptide of 170 amino acids. The deduced amino acid sequence of the sea perch TCTP gene showed a high similarity to that of zebrafish, rohu, rabbit, chicken and human. Sequence analysis revealed there were a signature sequence of TCTP family, an N-glycosylation site, and five Casein kinase phosphorylation sites in the sea perch TCTP. The temporal expression of TCTP genes in healthy and lipopolysaccharide (LPS) challenged fishes was measured by semi-quantitative reverse transcription-PCR (RT-PCR). The results indicated that LPS could up-regulate the expression of sea perch TCTP in the examined tissues, including head-kidney, spleen and liver.

Effect of lanthanum on expression of recombinant allophycocyanin gene in Pichia pastotis

The enhancing effect of lanthanum on gene expression of recombinant allophycocyanin (rAPC), a potential antitumor medicine, in Pichia pastoris was studied. PCR and sequence analysis were used in order to prove whether the APC gene had integrated into the yeast genome. The expression level of the recombinant allophycocyanin (rAPC) in BMMY medium containing LaCl3 was detected by ELISA method. The recombinant allophycocyanin was determined by Western blot. The results show that the recombinant Pichia pastoris chromosome contained allophycocyanin gene. Expression efficiency of rAPC gene in Pichia pastoris was promoted by proper LaCl3 concentration like 2, 5, 10 mmol (.) L-1, among which 5 mmol (.) L-1 was the most effective. The highest expression yield of rAPC in the BMMY medium containing 5 mmol (.) L-1 LaCl3 was 4.4 mg (.) L-1 at 48 h, that was increased by 110% compared with 2.1 mg (.) L-1 of control, in the meantime, the optimum culture time is shortened from 72 to 48 h. The result of western blot analysis indicates that the rAPC consisted of two kinds of subunits with molecular weight of 19 and 21 kDa respectively.