Molecular cloning and expression of a Relish gene in Chinese mitten crab Eriocheir sinensis

P>NF-kappa B is a B-cell specific transcription factor that plays crucial roles in inflammation, immunity, apoptosis, development and differentiation. In the present study, a novel NF-kappa B-like transcription factor Relish was cloned from Chinese mitten crab Eriocheir sinensis (designated as EsRelish) by rapid amplification of cDNA ends (RACE) technique based on expressed sequence tag (EST). The full-length cDNA of EsRelish was of 5034 bp, consisting of a 5' untranslated region (UTR) of 57 bp, a 3' UTR of 1335 bp with two mRNA instability motifs (ATTTA), a polyadenylation signal sequence (AATAAA) and a poly (A) tail, and an open reading frame (ORF) of 3645 bp encoding a polypeptide of 1214 amino acids with a calculated molecular mass of 134.8 kDa and a theoretical isoelectric point of 5.26. There were a typical Rel homology domain (RHD), two nuclear localization signal (NLS) sequences (KR), an inhibitor kappa B (I kappa B)-like domain with six ankyrin repeats, a PEST region and a death domain in the deduced amino acid sequence of EsRelish. Conserved domain, higher similarity with other Rel/NF-kappa Bs and phylogenetic analysis suggested that EsRelish was a member of the NF-kappa B family. Quantitative real-time RT-PCR was employed to detect the mRNA transcripts of EsRelish in different tissues and its temporal expression in hemocytes of E. sinensis challenged with Pichia methanolica and Listonella anguillarum. The EsRelish mRNA was found to be constitutively expressed in a wide range of tissues. It could be mainly detected in the hemocytes, gonad and hepatopancreas, and less degree in the gill, muscle and heart. The expression level of EsRelish mRNA in hemocytes was up-regulated from at 3, 6, 9 and 12 h after P. methanolica challenge. In L. anguillarum challenge, it was up-regulated at 9, 12 and 24 h. The results collectively indicated that EsRelish was potentially involved in the immune response against fungus and bacteria.

Cloning, characterization, and molecular application of a beta-agarase gene from Vibrio sp strain V134

V134, a marine isolate of the Vibrio genus, was found to produce a new beta-agarase of the GH16 family. The relevant agarase gene agaV was cloned from V134 and conditionally expressed in Escherichia coli. Enzyme activity analysis revealed that the optimum temperature and pH for the purified recombinant agarase were around 40 degrees C and 7.0. AgaV was demonstrated to be useful in two aspects: first, as an agarolytic enzyme, the purified recombinant AgaV could be employed in the recovery of DNA from agarose gels; second, as a secretion protein, AgaV was explored at the genetic level and used as a reporter in the construction of a secretion signal trap which proved to be a simple and efficient molecular tool for the selection of genes encoding secretion proteins from both gram-positive and gram-negative bacteria.

Cloning, characterization, and molecular application of a beta-agarase gene from Vibrio sp strain V134

V134, a marine isolate of the Vibrio genus, was found to produce a new beta-agarase of the GH16 family. The relevant agarase gene agaV was cloned from V134 and conditionally expressed in Escherichia coli. Enzyme activity analysis revealed that the optimum temperature and pH for the purified recombinant agarase were around 40 degrees C and 7.0. AgaV was demonstrated to be useful in two aspects: first, as an agarolytic enzyme, the purified recombinant AgaV could be employed in the recovery of DNA from agarose gels; second, as a secretion protein, AgaV was explored at the genetic level and used as a reporter in the construction of a secretion signal trap which proved to be a simple and efficient molecular tool for the selection of genes encoding secretion proteins from both gram-positive and gram-negative bacteria.

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.

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.

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.

Foreign gene transfer into Chinese shrimps (Penaeus chinensis) with gene gun

Plasmids pG DNA-RZ1 with a GFP (green fluorescent protein) reporter gene and a ribozyme gene incising penaeid white spot baculovirus (WSBV) were first introduced into the fertilized eggs of Chinese shrimps by gene gun. The treated and control samples of different development stages were observed with a fluorescent microscope. The transient expression of GFP gene was high in nauplius and zoea larvae. Results from RT-PCR and PCR for adults showed that the foreign genes had been transferred into the shrimps and had expressed the corresponding proteins. This work has established a transgenic method for penaeid shrimps, which will set base for the application of genetic engineering breeding into industry.

Construction of shuttle, expression vector of human tumor necrosis factor alpha (hTNF-alpha) gene and its expression in a cyanobacterium, Anabaena sp. PCC 7120

The construction of the shuttle, expression vector of human tumor necrosis factor alpha (hTNF-alpha) gene and its expression in a cyanobacterium Anabaena sp. PCC 7120 was reported. The 700-bp hTNF cDNA fragments have been recovered from plasmid pRL-rhTNF, then inserted downstream of the promoter PpsbA in the plasmid pRL439. The resultant intermediary plasmid pRL-TC has further been combined with the shuttle vector pDC-8 to get the shuttle, expression vector pDC-TNF. The expression of the rhTNF gene in Escherichia coil has been analyzed by SDS-PAGE and thin-layer scanning, and the results show that the expressed TNF protein with these two vectors is 16.9 percent (pRL-TC) and 15.0 percent (pDC-TNF) of the total proteins in the cells, respectively, while the expression level of TNF gene in plasmid pRL-rhTNF is only 11.8 percent. Combined with the participation of the conjugal and helper plasmids, pDC-TNF has been introduced into Anabaena sg PCC 7120 by triparental conjugative transfer, and the stable transgenic strains have been obtained. The existence of the introduced plasmid pDC-TNF in recombinant cyanobacterial cells has been demonstrated by the results of the agarose electrophoresis with the extracted plasmid samples and Southern blotting with alpha-(32)p labeled hTNF cDNA probes, while the expression of the hTNF gene in Anabaena sp. PCC 7120 has been confirmed by the results of Western blotting with extracted protein samples and human TNF-alpha monoclonal antibodies. The cytotoxicity assays using the mouse cancer cell line L929 proved the cytotoxicity of the TNF in the crude extracts from the transgenic cyanobacterium Anabaena sp. PCC 7120.

cDNA cloning, characterization and mRNA expression of a pacifastin light chain gene from the Chinese mitten crab Eriocheir sinensis

The pacifastin family, characterized by several conserved arrays of six cysteine residues, is a newly identified serine protease inhibitor (SPI) family discovered uniquely in arthropods and plays important roles in multiple biological processes. In the present study, the full-length cDNA of a pacifastin light chain (designated ESPLC) was cloned from the Chinese mitten crab Eriocheir sinensis by expressed sequence tags (ESTs) and PCR techniques. The 1036 bp ESPLC cDNA contained an 831 bp open reading frame (ORF) encoding a putative pacifastin-related peptide of 276 amino acids, a 5'-untranslated region (UTR) of 67 bp, and a 3'-UTR of 138 bp. Six putative conserved domains sharing a characteristic cysteine array (Cys-Xaa(9-12)-Cys-Asn-Xaa-Cys-Xaa-Cys-Xaa(2-3)-Gly-Xaa(3-4)-Cys-Thr-Xaa(3)-Cys) were identified in the deduced amino acid sequence of ESPLC. The conservation of these PLDs (pacifastin light chain domains) and the relative higher similarity of ESPLC to other pacifastin-related precursors suggested that ESPLC was a member of pacifastin family. The mRNA transcripts of ESPLC were found to be higher expressed in hepatopancreas, gill and haemolymph than in gonad, muscle and heart, with the highest expression level in hepatopancreas. The ESPLC mRNA expression in haemolymph of Chinese mitten crab was up-regulated at 2 h and 12 h after challenged with Listonella anguillarum. The tissue distribution and temporal characteristics of ESPLC mRNA expression, similar to that of prophenoloxidase gene in E. sinensis, suggested that ESPLC was potentially involved in the response against invading bacteria, with the possibility that it functioned in the prophenoloxidase system in E sinensis. (C) 2008 Elsevier Ltd. All rights reserved.

Molecular cloning, characterization and expression of heat shock protein 90 gene in the haemocytes of bay scallop Argopecten irradians

Heat shock protein 90 (HSP90) is a highly conserved molecular chaperone that plays key roles in the folding, maintenance of structural integrity and regulation of a subset of cytosolic proteins. In the present study, the cDNA of Argopecten irradians HSP90 (designated AiHSP90) was cloned by the combination of homology cloning and rapid amplification of cDNA ends (RACE) approaches. The full-length cDNA of AiHSP90 was of 2669 bp, including an open reading frame (ORF) of 2175 bp encoding a polypeptide of 724 amino acids with predicted molecular weight of 83.08 kDa and theoretical isoelectric point of 4.81. BLAST analysis revealed that AiHSP90 shared high similarity with other known HSP90s, and the five conserved amino acid blocks defined as HSP90 protein family signatures were also identified in AiHSP90, which indicated that AiHSP90 should be a cytosolic member of the HSP90 family. Fluorescent real-time quantitative PCR was employed to examine the expression pattern of AiHSP90 mRNA in haemocytes of scallops challenged by Gram-negative bacteria Vibrio anguillarum and Gram-positive bacteria Micrococcus luteus. In both bacterial challenged groups, the relative expression level of AiHSP90 transcript was up-regulated and reached maximal. level at 9 h after injection, and then dropped progressively to the original level at about 48 h post challenge. The results indicated that AiHSP90 was potentially involved in the immune responses against bacteria challenge in scallop A. irradian. (c) 2007 Elsevier Ltd. All rights reserved.

Molecular coordinated regulation of gene expression during ovarian development in the penaeid shrimp

To understand the molecular events of ovarian development in penaeid shrimp, RNA arbitrarily primed polymerase chain reaction (RAP-PCR) was used to identify differentially expressed genes during ovarian maturation in Metapenaeus ensis. From a screening of 700 clones in a cDNA library of the shrimp ovary by the products of RAP-PCR of different maturation stages, 91 fragments with differentially expressed pattern as revealed by dot-blot hybridization were isolated and sequenced. Forty-two of these fragments show significant sequence similarity to known gene products and the differentially expressed pattern of 10 putative genes were further characterized via Northern hybridization. Putative glyceraldehyde-3-phosphate dehydrogenase and arginine kinase are related to provision of energy for active cellular function in oocyte development. Translationally controlled tumor protein, actin, and keratin are related to the organization of cytoskeleton to accomplish growth and development of oocytes. High mobility group protein DSP1, heat shock protein 70, and nucleoside diphosphate kinase may act as repressors before the onset of ovarian maturation. Peptidyl-prolyl cis-trans isomerase and glutathione peroxidase are related to the stabilization of proteins and oocytes. This study provides new insights on the molecular events in the ovarian development in the shrimp.

Molecular cloning and immune responsive expression of a novel C-type lectin gene from bay scallop Argopecten irradians

C-type lectins are Ca2+-dependent carbohydrate-recognition proteins that play crucial roles in innate immunity. The cDNA of C-type lectin (AiCTL1) in the bay scallop Argopecten irradians was cloned by expressed sequence tag (EST) and RACE techniques. The full-length cDNA of AiCTL1 was 660 bp, consisting of a T-terminal. untranslated region (UTR) of 30 bp and a 3' UTR of 132 bp with a polyadenylation signal sequence AATAAA and a poly(A) tail. The AiCTL1 cDNA encoded a polypeptide of 166 amino acids with a putative signal peptide of 20 amino acid residues and a mature protein of 146 amino acids. The deduced amino acid sequence of AiCTL1 was highly similar to those of the C-type lectins from other animals and contained a typical carbohydrate-recognition domain (CRD) of 121 residues, which has four conserved disulfide-bonded cysteine residues that define the CRD and two additional cysteine residues at the amino terminus. AiCTL1 mRNA was dominantly expressed in the hemocytes of the bay scallop. The temporal expression of AiCTL1 mRNA in hemocytes was increased by 5.7-and 4.9-fold at 6 h after injury and 8 h after injection of bacteria, respectively. The structural features, high similarity and expression pattern of AiCTL1 indicate that the gene may be involved in injury heating and the immune response in A. irradians. (c) 2008 Elsevier Ltd. All rights reserved.

Molecular cloning, characterization and evolutionary analysis of phytoene desaturase (PDS) gene from Haematococcus pluvialis

Phytoene desaturase is one of the most important enzymes necessary for the biosynthesis of carotenoids in some cyanobacteria, green algae and plants. In this study, genomic DNA and cDNA of pds were cloned from unicellular green alga Haematococcus pluvialis strain323 using PCR and RT-PCR methods. The cDNA was cloned into plasmid pET-28a and efficiently expressed in Escherichia coli BL21. The complete genomic PDS gene of H. pluvialis, 3.3 kb in size, included eight exons and seven introns. To locate transcriptional regulation elements, an approximate 1 kb of 5'-flanking region was isolated by genome-walking method. Results of bioinformatic analysis showed several putative cis-elements e.g. the ABRE motif (abscisic acid responsive element), the C-repeat/DRE (dehydration responsive element) motif and the GCN4 motif were located in 5'-flanking region of pds. Results of phylogenetic analyses reveal that different sources of PDS genes form a separate clade, respectively, with 100% bootstrap support. Moreover, a maximum likelihood approach was employed to detect evidence of positive selection in the evolution of PDS genes. Results of branch-site model analysis suggest that 7.9% of sites along the green algal branch are under positive selection, and the PDS gene in green algae exhibits a different evolutionary pattern from its counterparts in cyanobacteria and plants.

The responsive expression of heat shock protein 22 gene in zhikong scallop Chlamys farreri against a bacterial challenge

HSP22 is a member of a small HSP subfamily contributing to the growth, transformation and apoptosis of the cell as well as acting as a molecular chaperone. In the present study, CfHSP22 cDNA was cloned from Chlamys farreri by the rapid amplification of cDNA ends technique. The full-length cDNA of CfHSP22 was of 1279 bp, consisting of a 5'-terminal untranslated region (5'UTR) of 122 bp, a 3'UTR of 581 bp with a canonical polyadenylation signal sequence AATAAA and a poly( A) tail, and an open reading frame of 576 bp encoding a polypeptide with a molecular mass of 22.21 kDa and a predicted isoelectric point of 9.69. There was an alpha-crystallin domain, a hallmark of the sHSP subfamily, in the C-terminus, and the deduced amino acid sequence of CfHSP22 showed high similarity to previously identified HSP22s. CfHSP22 was constitutively expressed in the haemocyte, muscle, kidney, gonad, gill, heart and hepatopancreas, and the expression level in the hepatopancreas was higher than that in the other tissues. CfHSP22 transcription was up-regulated and reached a maximal level at 12 h after the bacterial challenge, and then declined progressively to the original level at 48 h. These results suggested that CfHSP22 perhaps play a critical role in response to the bacterial challenge in haemocytes of scallop C. farreri.

Intracellular copper/zinc superoxide dismutase from bay scallop Argopecten irradians: Its gene structure, mRNA expression and recombinant protein

Superoxide dismutases are an ubiquitous family of enzymes that function to efficiently catalyze the dismutation of superoxide anions. Two unique and highly compartmentalized bay scallop Argopecten irradians superoxide dismutases: MnSOD and ecCuZnSOD, have been molecularly characterized in our previous study. To complete characterize the SOD family in A. irradians, a novel intracellular copper/zinc SOD from the A. irradians (Ai-icCuZnSOD) was obtained and characterized. The full-length cDNA of Ai-icCuZnSOD was 1047 bp with a 459 bp open reading frame encoding 152 amino acids. The genomic length of the Ai-icCuZnSOD gene was about 4279 bp containing 4 exons and 3 introns. The promoter region containing many putative transcription factor binding sites were analyzed. Furthermore, quantitative reverse transcriptase real-time PCR (qRT-PCR) analysis indicated that the highest expression of the Ai-icCuZnSOD was detected in gill and the expression profiles in hemocytes of bay scallops challenged with bacteria Vibrio anguillarum and lipopolysaccharide (LPS) were different. The result presented an increased expression after injection with LPS whereas no significant changes were observed after V. anguillarum injection. A fusion protein containing Ai-icCuZnSOD was produced in vitro. The rAi-icCuZnSOD is a stable enzyme, retaining more than 80% of its activity between 10 and 60 degrees C and keeping above 88% of its activity at pH values between 5.8 and 9. Ai-icCuZnSOD is more stable under alkaline than acidic conditions. Crown Copyright (C) 2009 Published by Elsevier Ltd. All rights reserved.