Isolation and characterization of Toll-like receptor 9 in half-smooth tongue sole Cynoglossus semilaevis

Toll-like receptors (TLRs) are considered as key sensors to trigger the host's innate immune system and adaptive immune responses by recognizing various PAMPs and initiating signal transduction. TLR9, as a member of TLR family, mediates the recognition of unmethylated CpG dinucleotide motifs commonly found in both bacterial and viral genomes. In the current study, the TLR9 gene was isolated from one of flatfish species, half-smooth tongue sole (Cynoglossus semilaevis). In the 4588 bp genomic sequence, three exons, two introns, and 5′ UTR of 23 bp and 3′ UTR of 342 bp were identified. Putative amino acid sequence was 1062 residues long, including a typical conserved cytosolic Toll/interleukin-1 receptor (TIR) domain, 14 leucine-rich repeat (LRR) motifs, with greater than 60% identity to gilthead sea bream Sparus aurata and Japanese flounder Paralichthys olivaceus orthologs. Quantitative RT-PCR analysis indicated a broad expression of csTLR9, especially in spleen and gonads. No statistically significant changes were observed for csTLR9 mRNA levels in spleen and head kidney after inactive Vibrio anguillarum immunisation. In C. semilaevis ontogeny, the expression of csTLR9 appeared to be developmentally regulated. The presence of maternal TLR9 mRNA and the dramatic decrease of TLR9 expression at metamorphic stage indicated TLR9 might be involved in C. semilaevis development. Comparing sequence and expression profile of csTLR9 with mammalian and other piscine TLR9s suggested that the main function of TLR9 might be conserved across vertebrates, although species-specific features were present.     

Identification and characterization of IL-1 receptor-associated kinase-4 (IRAK-4) in half-smooth tongue sole Cynoglossus semilaevis

As a crucial component in TLR/IL-1R signaling pathways, IRAK-4 plays a central role in innate and adaptive immunity. In the present study, the cDNA of IRAK-4 was cloned for the first time from half-smooth tongue sole (Cynoglossus semilaevis). The full-length cDNA of csIRAK-4 was 2149 bp and contained a 168 bp 5′ UTR, a 580 bp 3′ UTR and a 1401 bp CDS. The predicted protein sequence of csIRAK-4 had two typical domains, a death domain (DD) at the N terminus and a serine/threonine/tyrosine protein kinase domain (STYKc) at the C terminus. RT-PCR showed that csIRAK-4 mRNA was detected in all tested tissues, especially in immune-related organs, gonads and brain. After injected with inactivated Vibrio anguillarum, the expressions of csIRAK-4 were up-regulated significantly (P < 0.05) in spleen and head kidney. During development, csIRAK-4 was expressed at all selected stages and low-level expression was detected at metamorphosis. Taken together, the present study indicated that csIRAK-4 played a crucial role in immune responses and might be involved in the process of development.     

cDNA cloning, characterization and expression analysis of peroxiredoxin 5 gene in the ridgetail white prawn Exopalaemon carinicauda

Peroxiredoxin is a superfamily of antioxidative proteins that play important roles in protecting organisms against the toxicity of reactive oxygen species. In this study, a full-length of peroxiredoxin 5 (designated EcPrx5) cDNA was cloned from the ridgetail white prawn Exopalaemon carinicauda by using rapid amplification of cDNA ends (RACE) approaches. The full-length cDNA of the EcPrx5 was of 827 bp, containing a 5′ untranslated region (UTR) of 14 bp, a 3′ UTR of 228 bp with a poly (A) tail, and an open reading frame of 585 bp encoding a polypeptide of 194 amino acids with the predicted molecular weight of 20.83 kDa and estimated isoelectric point of 7.62. BLAST analysis revealed that amino acids of EcPrx5 shared 89, 68, 66, 65, 53 and 51 % identity with that of Macrobrachium rosenbergii, Megachile rotundata, Harpegnathos saltator, Acromyrmex echinatior, Danio rerio, and Homo sapiens counterparts, respectively. The conserved Prx domain and the signature of peroxiredoxin catalytic center identified in EcPrx5 suggested that EcPrx5 belonged to the atypical 2-Cys Prx subgroup. Real time quantitative RT-PCR analysis indicated that EcPrx5 could be detected in all the tested tissues with highest expression level in hepatopancreas. As time progressed, the expression level of EcPrx5 both in hemocytes and hepatopancreas increased in the first 6 h after Vibrio anguillarum and white spot syndrome virus challenge, and showed different expression profiles. The results indicated that EcPrx5 involved in immune response against bacterial and viral infection in E. carinicauda.     

Molecular characterization and expression analysis of a KIFC1-like kinesin gene in the testis of Eumeces chinensis

The member of the kinesin-14 subfamily, KIFC1, is a carboxyl-terminal motor protein that plays an important role in the elongation of nucleus and acrosome biogenesis during the spermiogenesis of mammals. Here, we had cloned and sequenced the cDNA of a mammalian KIFC1 homologue (termed ec-KIFC1) from the total RNA of the testis of the reptile Eumeces chinensis. The full-length sequence was 2,339 bp that contained a 216 bp 5′-untranslated region (5′UTR), a 194 bp 3′-untranslated region (3′UTR) and a 1,929 bp open reading frame that encoded a special protein of 643 amino acids (aa). The calculated molecular weight of the putative ec-KIFC1 was 71 kDa and its estimated isoelectric point was 9.47. The putative ec-KIFC1 protein owns a tail domain from 1 to 116 aa, a stalk domain from 117 to 291 aa and a conserved carboxyl motor domain from 292 to 642 aa. Protein alignment demonstrated that ec-KIFC1 had 45.6, 42.8, 44.6, 36.9, 43.7, 46.4, 45.1, 55.6 and 49.8 % identity with its homologues in Mus musculus, Salmo salar, Danio rerio, Eriocheir sinensis, Rattus norvegicus, Homo sapiens, Bos taurus, Gallus gallus and Xenopus laevis, respectively. Tissue expression analysis showed the presence of ovary, heart, liver, intestine, oviduct, testis and muscle. The phylogenetic tree revealed that ec-KIFC1 was more closely related to vertebrate KIFC1 than to invertebrate KIFC1. In situ hybridization showed that the ec-KIFC1 mRNA was localized in the periphery of the nuclear membrane and the center of the nucleus in early spermatids. In mid spermatids, the ec-KIFC1 had abundant expression in the center of nucleus, and was expressed in the tail and the anterior part of spermatids. In the late spermatid, the nucleus gradually became elongated, and the ec-KIFC1 mRNA signal was still centralized in the nucleus. In mature spermatids, the signal of the ec-KIFC1 gradually became weak, and was mainly located at the tail of spermatids. Therefore, the ec-KIFC1 probably plays a critical role in the spermatogenesis of E. chinensis.     

The co-existence of two growth hormone receptors and their differential expression profiles between female and male tongue sole (Cynoglossus semilaevis)

Growth hormone receptor (Ghr) is a single-transmembrane pass protein which is important in initiating the ability of growth hormone (Gh) to regulate development and somatic growth in vertebrates. In this study, molecular cloning, expression analysis of two different ghr genes (ghr1 and ghr2) in the tongue sole (Cynoglossus semilaevis) was conducted. As a result, the ghr1 and ghr2 cDNA sequences are 2364 bp and 3125 bp, each of which encodes a transmembrane protein of 633 and 561 amino acids (aa), respectively. Besides, the ghr1 gene includes nine exons and eight introns. The sex-specific tissue expression was analyzed by using 14 tissues from females, normal males and extra-large male adults. Both the ghr1 and ghr2 were predominantly expressed in the liver, and the ghr1 expression level in normal males was 1.6 and 1.4 times as much as those in females and extra-large males, while the ghr2 mRNA expression level in normal males was 1.1 and 1.2 times as much as those in females and extra-large males, respectively. Ontogenetic expression analysis at early life stages indicated that the ghr1 and ghr2 mRNAs were detected at all of the 35 sampling points (from oosphere to 410 days-old). Furthermore, the sex differences in ghr mRNA expressions were also examined by using a full-sib family of C. semilaevis. Significantly higher levels of ghr1 mRNA were observed in males than in females at most stages of the sampling period (P < 0.01). The ghr2 mRNA expression at most stages exhibited a significant sexual difference at each sampling point (P < 0.01) without any variation trend related with the sexes during the whole sampling period.     

Molecular cloning, characterization and expression of cDNA encoding translationally controlled tumor protein (TCTP) from Jatropha curcas L

A cDNA encoding translationally controlled tumor protein (TCTP) of Jatropha curcas L., JcTCTP, was isolated from an endosperm cDNA library. JcTCTP consisted of a 5?? untranslated region (UTR) of 526 bp, a 3?? UTR of 377 bp and an open reading frame (ORF) of 507 bp, encoding a protein of 168 amino acid residues, which contained two signature sequences of TCTP family. Its deduced amino acid sequence was similar to the other known plants TCTPs in a range of 77.4?C92.3%. Expression of JcTCTP was the highest in the stem, endosperm at embryo formation stage and embryo of J. curcas tissues, and the lowest in the endosperm at seminal leaf embryo stage and flower, demonstrating a pattern of temporal and spatial specific expression.     

cDNA cloning, tissues, embryos and larvae expression analysis of Sox10 in half-smooth tongue-sole, Cynoglossus semilaevis

A half-smooth tongue-sole, Cynoglossus semilaevis Sox10 (Accession no.: EU070763) was isolated from brain of tongue sole by using homologous cloning and RACE method. The complete cDNA of the tongue sole Sox10 contains a 35 bp 5′UTR, a 1338 bp open reading frame (ORF) encoding 445 amino acids and a 1155 bp 3′UTR. A condensed phylogenetic tree was constructed based on the amino acid sequences of tongue sole Sox10 and other well-defined vertebrate Sox. The overall topology of the tree showed the tongue sole Sox10 clusters with all Sox10. Alignment of amino acid residues of the tongue sole Sox10 gene with those from other vertebrate indicated high level conservation of amino acid sequence. The RT-PCR analysis demonstrated that the tongue sole Sox10 was highly expressed in brain, gills, skin and eyes, intermediately in spleen, heart, head–kidney and muscles, weakly expressed in kidneys and intestine and no expression in liver and gonad. The Sox10 was also expressed weakly in germ cell and zygote. We cannot detect the expression of the Sox10 in 8-cells stage. However it resumed expression weakly from blastula stage to middle of gastrula. And it expressed highly from neurula stage to 25 dah (day after hatching). It suggested that the Sox10 was involved in the development of embryos and larvae in tongue sole.     

Molecular cloning, expression analysis of insulin-like growth factor I (IGF-I) gene and IGF-I serum concentration in female and male Tongue sole (Cynoglossus semilaevis)

Insulin-like growth factor I (IGF-I) is a polypeptide hormone that regulates growth during all stages of development in vertebrates. To examine the mechanisms of the sexual growth dimorphism in the Tongue sole (Cynoglossus semilaevis), molecular cloning, expression analysis of IGF-I gene and IGF-I serum concentration analysis were performed. As a result, the IGF-I cDNA sequence is 911 bp, which contains an open reading frame (ORF) of 564 bp encoding a protein of 187 amino acids. The sex-specific tissue expression was analyzed by using 14 tissues from females, normal males and extra-large male adults. The IGF-I mRNA was predominantly expressed in liver, and the IGF-I expression levels in females and extra-large males were 1.9 and 10.2 times as much as those in normal males, respectively. Sex differences in IGF-I mRNA expressions at early life stages were also examined by using a full-sib family of C. semilaevis, and the IGF-I mRNA was detected at all of the 27 sampling points from 10 to 410 days old. An increase in IGF-I mRNA was detected after 190 day old fish. The significantly higher levels of IGF-I mRNA in females were observed after 190 days old in comparison with males (P < 0.01). The IGF-I concentrations in serum of mature individuals were detected by ELISA. The IGF-I level in the serum of females was approximately two times as much as that of males. Consequently, IGF-I may play an important role in the endocrine regulation of the sexually dimorphic growth of C. semilaevis.     

Molecular cloning and expression of a C-type lectin gene from Venerupis philippinarum

C-type lectins have been demonstrated to play important roles in invertebrate innate immunity by mediating the recognition of pathogens and clearing the micro-invaders. In the present study, a C-type lectin gene (denoted as VpCTL) was identified from Venerupis philippinarum by expressed sequence tag and rapid amplification of cDNA ends approaches. The full-length cDNA of VpCTL consists of 904 nucleotides with an open-reading frame of 456 bp encoding a peptide of 151 amino acids. The deduced amino acid sequence of VpCTL shared high similarity with C-type lectins from other species. The C-type lectin domain and the characteristic EPN and WND motifs were found in VpCTL. The VpCTL mRNA was dominantly expressed in the haemocytes of the V. philippinarum. After Listonella anguillarum challenge, the temporal expression of VpCTL mRNA in haemocytes was increased by 97- and 84-fold at 48 and 96 h, respectively. With high expression level in haemocytes and hepatopancreas, and the up-regulated expression in haemocytes indicted that VpCTL was perhaps involved in the immune responses to L. anguillarum challenge.     

Identification and expression of the elongator protein 2 (Ajelp2) gene,a novel regeneration-related gene from the sea cucumber Apostichopus japonicus

Elongator proteins comprise six subunits (ELP1–ELP6) and form protein complexes. The elongator protein 2 gene (elp2) encodes a protein with a WD40 repeats domain that acts as a scaffold for complex assembly. It also plays an important role in growth and development. In this study, the full-length cDNA of elongator protein 2 (Ajelp2) was cloned from the sea cucumber Apostichopus japonicus (A. japonicus) using rapid amplification of cDNA ends PCR techniques and comprised 3,058 bp, including a 54 bp 5′ untranslated (UTR), a 526 bp 3′ UTR and a 2,478 bp open reading frame encoding a polypeptide of 825 amino acids. The Ajelp2 sequence showed high homology to 12 other species. The molecular weight and isoelectric of point the presumptive protein were 91.6 kDa and 5.84, respectively. In situ hybridization indicated that the gene is expressed in the body wall, intestine, respiratory tree and longitudinal muscle. The expression level of Ajelp2 increased in recovering of organs in sea cucumber and showed it’s the highest expression level at the 15th day in the intestine and respiratory tree. Its expression then gradually decreased to normal levels. In the body wall, the expression level of Ajelp2 was up-regulated and then down-regulated. These results indicated that Ajelp2 is involved in protein regulation during the regeneration process in the sea cucumber A. japonicus.     

Molecular cloning,characterisation and expression of the translationally controlled tumor protein gene in rock bream (Oplegnathus fasciatus)

Translationally controlled tumor protein (TCTP) is an important immune regulator that has been implicated in a number of cellular processes, including cell growth, cell cycle progression, apoptosis regulation and protection of cells against various environmental stresses. In this study, we cloned and characterised TCTP from rock bream (Oplegnathus fasciatus), which is an economically important species in the Korean aquaculture industry. The full-length rock bream TCTP (RbTCTP) cDNA was of 1,041 bp and contained an open reading frame of 513 bp, which encoded 170 amino acids. The 5′ untranslated region (UTR) was 90 bp, while the 3′ UTR was 438 bp, containing a polyadenylation signal. RbTCTP showed 76, 75 and 74 % amino acid sequence identities to those of tilapia (Oreochromis niloticus), orange-spotted grouper (Epinephelus coioides) and Japanese sea perch (Lateolabrax japonicus), respectively. The positions of microtubule binding region, Ca⁺ binding region and TCTP signature regions in RbTCTP were similar to other fish species and mammals. RbTCTP mRNA expression level was highest in the gill compared to other tissues. The level of RbTCTP mRNA expression was significantly regulated by injection of red seabream iridovirus, Streptococcus iniae and Edwardsiella tarda.     

Population genetic structure of the tongue sole (Cynoglossus semilaevis) in Korea based on multiplex PCR assays with 12 polymorphic microsatellite markers

The tongue sole, Cynoglossus semilaevis, is an important fishery resource in Korea. About 100 tongue sole sampled from three major habitats along the western coast of Korea were assessed using multiplex assays with 12 highly polymorphic microsatellite loci to explore the population genetic structure of the species; 151 alleles and similar high levels of gene diversity (mean number of alleles (N_A) = 10.42, mean expected heterozygosity (He) = 0.78) were detected. Three populations showed significant Hardy–Weinberg equilibrium deviations at four loci. Although a significant difference in the number of unique alleles was observed among populations, genetic population subdivision was low by F-statistics (overall F _ST = 0.007, p < 0.05). However, this substructure was not supported by analysis of molecular variance or analyses of isolation by distance. The results suggest a lack of genetic structure among the tongue sole populations in Korean waters and that the populations should be managed as a single unit. The lack of genetic differentiation among samples may be due to high levels of larval dispersal in ocean currents. Alternatively, the populations may have diverged too recently for significant genetic differentiation to have become evident. Given the intensity of tongue sole aquaculture activity in China, which adjoins the western coast of Korea, the possibility that aquaculture may have partially contributed to the population genetic characteristics detected cannot be excluded. This study provides the basic information on nature population structure of C. semilaevis that may help to preserve and manage tongue soles in Korea.