Genes In Large Yellow Croaker Research Articles

The identification of toll-like receptor genes in large yellow croaker (Larimichthys crocea): provides insights into its environmental adaptation to biological and abiotic stresses

The identification of toll-like receptor genes in large yellow croaker (Larimichthys crocea): provides insights into its environmental adaptation to biological and abiotic stresses

Transcriptome analysis revealed changes in multiple genes in Larimichthys crocea under starvation stress

To identify important functional genes of the large yellow croaker (Larimichthys crocea) under starvation stress, the expression changes of genes in muscles tissue of fish fasted for 0, 4, 8, 12, 16, and 20 d were analyzed with RNA-seq technology. Sequencing results showed that a total of 507 million clean reads were generated. Totals of 662, 998, 1015, 1260, and 1122 of differentially expressed genes (DEGs) were obtained at 4, 8, 12, 16, and 20 d, respectively, using starvation 0 d as control. Gene ontology (GO) classification and analysis showed that DEGs could be classified into 42 categories of biological processes, cellular components, and molecular functions. Of these, DEGs are enriched in regulations such as metabolic process, cellular process and single-organism process. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that DEGs involved a total of 22 pathways, eight of which were metabolic pathways. With fewer pathways enrichment entry in early starvation stages, but more pathways enrichment entry in late starvation stages (such as at 16 days of starvation), DEGs were significantly enriched in glycolysis/gluconeogenesis, biosynthesis of amino acids, carbon metabolism, fructose and mannose metabolism, methane metabolism and carbon fixation in photosynthetic organisms. After 20 days of starvation, DEGs began to be significantly enriched in the immune pathway, indicating that at this time, fish began to show an immunomodulatory response. This study systematically disclosed the changes of muscle tissue genes in large yellow croaker under starvation stress. The obtained results contribute to research on environmental stress in teleost fish.

Transcriptome and methylome dynamics in the gills of large yellow croaker (Larimichthys crocea) during low-salinity adaption

DNA methylation is a critical epigenetic modification that dynamically regulates gene expression in organisms facing abiotic stress. However, few studies have comprehensively examined the role of DNA methylation in marine fish during environmental adaptation. Therefore, this study explored the methylome dynamics and DNA methylation regulation mechanisms in large yellow croaker (Larimichthys crocea) during low-salinity adaption. The methylation level in the gills was notably raised in the S-group (5‰ salinity) compared to C-group (25‰ salinity). A total of 109 differentially methylated promoter target genes and 581 differentially expressed genes were identified via whole-genome bisulfite sequencing (WGBS) and RNA-seq of gills in the two salinity groups, respectively. Moreover, 23 hypo-methylated/up-regulated differentially methylated genes (DMGs) and 28 hyper-methylated/down-regulated DMGs were identified through integrative analysis, which were mainly enriched in signal transduction, ion exchange, energy metabolism, and cytoskeleton system and other biological processes. Collectively, our findings suggested that low-salinity stress can induce adaptive genome-wide DNA methylation changes, which can in turn affect the transcription of genes in large yellow croaker during low-salinity adaptation. Therefore, our findings provide new insights into the regulatory mechanisms of marine fish in response to rapid environmental changes.

Dual RNA-seq reveals a host-pathogen interaction transcriptional regulation pattern between Cryptocaryon irritans and large yellow croaker (Larimichthys crocea)

Cryptocaryon irritans is an obligate ectoparasite of marine fish that causes severe damage to wild and farmed marine fish populations due to its wide parasitic range. Recently, the publication of the draft genome of C. irritans provides the necessary genetic basis for studying virulence mechanisms. In this study, the marine economic fish large yellow croaker and C. irritans were used as models to identify 5520 and 700 differentially expressed genes (DEGs) by dual RNA-seq technology with high sequencing depth (12×), revealing the dynamic expression patterns of host-pathogen interaction after infection. The results of functional enrichment showed that during C. irritans infection, the enhancement of metabolism and activation of immune regulatory pathways were enriched in large yellow croaker, while the increased proliferation and synthesis of surface antigens was enriched in C. irritans. Protein-protein interaction (PPI) network analysis identified 10 hub genes in large yellow croaker (myc, becn1, tbk1, stat1, stat3, irf3, hif1α, myd88, cdc42, and syk) and 2 hub genes in C. irritans (mcm7 and cak76459). In addition, further correlation studies showed that hif1α and mcm7 were interacting gene pairs significantly related to the infection process. This study is helpful in exploring the molecular regulation mechanism of large yellow croaker after C. irritans infection and provides a necessary theoretical basis for the upcoming host-pathogen interaction research.

Effects of low-salinity acclimation on the Na+/K+ ATPase activity and expression of osmoregulatory-related genes in large yellow croaker (Larimichthys crocea)

Large yellow croaker (Larimichthys crocea) is one of the most commercially important marine fish in southeast China. The white spot disease frequently occurs in large yellow croaker leading to a devastating loss. Given that the pathogen, Cryptocaryon irritans, hardly survive in the low-salinity environment, the low-salinity farming is a potential strategy to defend large yellow croaker. In this study, the water salinity was consecutively decreased from 28 ‰ to 0 ‰, during which the survival rate of L. crocea was stable from 28 ‰ to 4 ‰, while a higher mortality was observed at < 4 ‰. The NKA activity in the kidney and gill of L. crocea was significantly stimulated by the decrease of water salinity from 28 ‰ to 4 ‰ and gradually dropped to the baseline, which was associated with variations of the expression level of NKAα1 and NKAβ1. The expression profile of PRLR was consistent with NKAα1 and NKAβ1, which implied that they might be the targets of PRL in L. crocea in response to the challenge of salinity variations. The other two osmoregulatory-related genes, NHEβ and NCC, significantly increased at low water salinity (< 4 ‰) in both the kidney and gill, which likely dedicated the sodium reabsorption at low water salinity. Our findings would provide new insights into osmoregulation of the kidney and gill in the large yellow croaker in response to the challenge of lowing water salinity.

Effects of salinity stress on methylation of the liver genome and complement gene in large yellow croaker (Larimichthys crocea)

Salinity is an important environmental factor that affects the yield and quality of large yellow croaker (Larimichthys crocea) during aquaculture. Here, whole-genome bisulfite sequencing (WGBS), RNA-seq, bisulfite sequencing PCR (BSP), quantitative real-time PCR (qPCR), and dual luciferase reporter gene detection technologies were used to analyze the DNA methylation characteristics and patterns of the liver genome, the expression and methylation levels of important immune genes in large yellow croaker in response to salinity stress. The results of WGBS showed that the cytosine methylation of CG type was dominant, CpGIsland and repeat regions were important regions where DNA methylation occurred, and the DNA methylation in upstream 2k (2000bp upstream of the promoter) and repeat regions had different changes in the liver tissue of large yellow croaker in the response to the 12‰, 24‰, 36‰ salinity stress of 4 w (weeks). In the combined analysis of WGBS and transcriptome, the complement and coagulation cascade pathways were significantly enriched, in which the complement-related genes C7, C3, C5, C4, C1R, MASP1, and CD59 were mainly changed in response to salinity stress. In the studied area of MASP1 gene promoter, the methylation levels of many CpG sites as well as total cytosine were strongly negatively correlated with mRNA expression level. Methylation function analysis of MASP1 promoter further proved that DNA methylation could inhibit the activity of MASP1 promoter, indicating that salinity may affect the expressions of complement-related genes by DNA methylation of gene promoter region.

Characterization of Caspase8 and its role in the regulation of apoptosis-related genes in large yellow croaker (Larimichthys crocea)

The structure and function of Caspase8, a vital initiator caspase in apoptosis have been extensively studied in different species. However, the role of Caspase8 in Larimichthys crocea remains unknown. Therefore, the main aim of this study was to characterize Caspase8 in Larimichthys crocea and explore its role in the regulation of apoptosis-related genes. In this study, we molecularly characterized the full-length coding sequence of Caspase8 and investigated the role of Caspase8 in the regulation of apoptosis-related genes using qRT-PCR and western blot in Larimichthys crocea. Our results demonstrated that the gene and protein expression levels of Caspase8 were upregulated upon treatment with increasing levels of caspase activator (hydrogen peroxide, H2O2). Furthermore, activation of Caspase8 by birinapant significantly increased the mRNA expression of apoptosis-related genes, and this effect was blocked by the knockdown of Caspase8 in hepatocytes. Also, palmitic acid (PA) and linoleic acid (LA) upregulated the transcription of apoptosis-related genes, but only PA induced apoptosis through Caspase8 in vivo and in vitro. Taken together, the findings of the present study will promote further exploration into the structure and function of Larimichthys crocea Caspase8 at the molecular level and indicate that the apoptosis system may be regulated by different fatty acids.

Effects of High Levels of Dietary Linseed Oil on the Growth Performance, Antioxidant Capacity, Hepatic Lipid Metabolism, and Expression of Inflammatory Genes in Large Yellow Croaker (Larimichthys crocea).

A growth experiment was conducted to evaluate the effects of dietary fish oil (FO) replaced by linseed oil (LO) on the growth performance, antioxidant capacity, hepatic lipid metabolism, and expression of inflammatory genes in large yellow croaker (Larimichthys crocea). Fish (initial weight: 15.88 ± 0.14 g) were fed four experimental diets with 0% (the control), 33.3%, 66.7%, and 100% of FO replaced by LO. Each diet was randomly attributed to triplicate seawater floating cages (1.0 × 1.0 × 2.0 m) with 60 fish in each cage. Results showed that the growth performance of fish fed the diet with 100% LO was markedly decreased compared with the control group (P < 0.05), while no remarkable difference was observed in the growth performance of fish fed diets within 66.7% LO (P > 0.05). The percentage of 18:3n-3 was the highest in the liver and muscle of fish fed the diet with 100% LO among the four treatments. When dietary FO was entirely replaced by LO, fish had a markedly higher total cholesterol, total triglyceride, low-density lipoprotein cholesterol content, and alanine transaminase activity in the serum than the control group (P < 0.05). The concentration of malondialdehyde was markedly higher, while the activity of catalase was markedly lower in fish fed the diet with 100% LO than the control group (P < 0.05). When dietary FO was entirely replaced by LO, hepatic lipid content, transcriptional levels of fatp1 and cd36, and CD36 protein expression were significantly higher, while transcriptional level of cpt-1 and CPT-1 protein expression were significantly lower than the control group (P < 0.05). As for the gene expression of cytokines, fish fed the diet with 100% LO had markedly higher transcriptional levels of il-1β, tnfα, and il-6 than the control group (P < 0.05). In conclusion, the substitution of 66.7% FO with LO had no significant effects on the growth performance of fish, while 100% LO decreased the growth performance and increased the inflammation and hepatic lipid content of fish. The increase of hepatic lipid content was probably due to the increased fatty acid uptake and decreased fatty acid oxidation in fish.

Effects of dietary curcumin on growth, antioxidant capacity, fatty acid composition and expression of lipid metabolism-related genes of large yellow croaker fed a high-fat diet.

A 10-week feeding trial was conducted to investigate the effect of dietary curcumin (CC) on growth antioxidant responses, fatty acid composition, and expression of lipid metabolism-related genes of large yellow croaker fed a high-fat diet (HFD). Four diets (lipid level at 18 %) were formulated with different levels of curcumin (0, 0·02, 0·04 and 0·06 %). The best growth performance was found in the 0·04 % curcumin group, with the body and hepatic lipid levels lower than the control group (0 % CC). The content of TAG, total cholesterol and LDL-cholesterol was the least in the 0·06 % curcumin group. The lowest malondialdehyde and the highest superoxide dismutase, catalase and total antioxidant capacity were observed in the 0·04 % curcumin group. The 0·04 % curcumin group had higher expression of Δ6fad, elovl5 and elovl4 and showed higher hepatic n-6 and n-3 PUFA. Expression of ppara, cpt1, and aco was significantly increased, while expression of srebp1 and fas was dramatically decreased in curcumin groups compared with the control group. Overall, 0·04 % curcumin supplementation could mitigate the negative effects caused by HFD and promote growth via reducing hepatic lipid deposition, improving antioxidant activity and increasing PUFA of large yellow croaker. To conclude, abnormal hepatic lipid deposition was probably due to increased fatty acid oxidation and reduced de novo synthesis of fatty acids.

Effects of dietary terrestrial oils supplemented with L-carnitine on growth, antioxidant capacity, lipid metabolism and inflammation in large yellow croaker (Larimichthys crocea).

The present study was conducted to determine the effects of dietary terrestrial oils (TO) supplemented with L-carnitine on growth performance, biochemical and antioxidant response, lipid metabolism and inflammation in large yellow croaker (Larimichthys crocea). Three iso-nitrogenous and iso-lipidic experimental diets were formulated with FO (fish oil, the control group), 75% TO (75% FO was substituted by the oil mixture with equal amounts of soybean oil, linseed oil and pork lard) and 75% TOC (75% TO supplemented with 800 mg/kg L-carnitine), respectively. Compared to the control group, feed efficiency ratio and specific growth rate were significantly increased in fish fed diets with 75% TO and 75% TOC. Hepatic lipid content, serum triglyceride level, low-density lipoprotein-cholesterol level and the mRNA expression of pro-inflammatory genes (tnfα and ifnγ) were significantly increased in fish fed the diet with 75% TO compared to the control group. However, the supplementation of 800 mg/kg L-carnitine in the 75% TO diet repressed hepatic lipid content, serum low-density lipoprotein-cholesterol level and the mRNA expression of tnfα and ifnγ in fish compared to fish fed the diet with 75% TO. Total antioxidant capacity, the activity of superoxide dismutase, the mRNA expression of cpt-I and the activity of CPT-I were significantly increased in fish fed the diet with 75% TOC compared to 75% TO. In conclusion, these results suggested that the supplementation of 800 mg/kg L-carnitine in the diet with terrestrial oils mixture could increase growth, antioxidant capacity, fatty acid oxidation and decrease the expression of inflammatory genes in large yellow croaker.

Genome-wide investigation of Dmrt gene family in large yellow croaker (Larimichthys crocea)

The Dmrt (Doublesex and Mab-3 related transcription factor) gene family is a class of crucial transcription factors characterized by a conserved DM (Doublesex/Mab-3) domain. Previous researches indicate this gene family is involved in various physiological processes, especially in sex determination/differentiation and gonad development. Despite the vital roles of the Dmrt gene family in physiological processes, the comprehensive characterization and analysis of the dmrt genes in large yellow croaker (Larimichthys crocea), one of the most commercially important marine fish in China, have not been described. In this study, we performed the first genome-wide systematic analysis of L. crocea dmrt genes through the bioinformatics method. A total of seven members of the Dmrt gene family including Lcdmrt1, Lcdmrt2a, Lcdmrt2b, Lcdmrt3, Lcdmrt4, Lcdmrt5, and Lcdmrt6 were excavated based on the genome data of L. crocea. Further analysis revealed that the dmrt genes of L. crocea were distributed unevenly across four chromosomes. There were three dmrt genes (Lcdmrt1, Lcdmrt2a, and Lcdmrt3) on 3rd chromosome, one (Lcdmrt6) on 13th chromosome, one (Lcdmrt4) on 14th chromosome, two on (Lcdmrt5 and Lcdmrt2b) 17th chromosome. The gene structure analysis indicated that the number of introns of different dmrt genes of L. crocea had some differences: Lcdmrt1 had four introns, Lcdmrt2a, Lcdmrt2b, and Lcdmrt6 had two introns, Lcdmrt3, Lcdmrt4, and Lcdmrt5 had only one intron. The expression pattern analysis with published gonad transcriptome datasets and further confirmed by qRT-PCR revealed that these members of the Dmrt gene family except for Lcdmrt4 were all sexually dimorphic and preferred expressing in testis. Furthermore, the expression pattern analysis also revealed that the expression level of Lcdmrt1 and Lcdmrt6 was significantly higher than that of other members, suggesting that these two genes may play a more important role in testis. Overall, our studies provide a comprehensive insight into the Dmrt gene family members and a basis for the further study of their biological functions in L. crocea.

Effects of dietary tea polyphenols on growth, biochemical and antioxidant responses, fatty acid composition and expression of lipid metabolism related genes of large yellow croaker(Larimichthys crocea)

The study was conducted to investigate the effects of dietary tea polyphenols (TP) on growth performance, biochemical and antioxidants responses, fatty acid composition, and lipid metabolism-related gene expressions of large yellow croaker (Larimichthys crocea). Four diets were formulated with different levels of TP (0.00%, 0.01%, 0.02% and 0.05%). Results showed that growth performance of L. crocea were not different among dietary treatments. Compared with the control group, fish in 0.02% TP group had lower body and hepatic lipid content and lower total cholesterol content. The minimum content of triglycerides and low-density lipoprotein-cholesterol were found in 0.05% TP group. Hepatic n-6 PUFA and n-3 PUFA were significantly higher in TP supplementation groups. Malondialdehyde content was lower in TP supplementation groups, and superoxide dismutase activity was higher in 0.01% TP group than the control group. The mRNA expressions of carnitine palmitoyltransferase1, acyl-CoA oxidase and peroxisome proliferators-activated receptor α were up-regulated in 0.01% and 0.02% TP groups, while lipoprotein lipase expression was down-regulated in TP supplementation groups than the control group. Results suggested that 0.01%–0.02% TP supplementation could reduce the deposition of liver lipid of L. crocea caused by high-lipid diet, which might be due to the increase in lipid oxidation related gene expressions.

Molecular characterization and expression analyses of three RIG-I-like receptor signaling pathway genes (MDA5, LGP2 and MAVS) in Larimichthys crocea.

In this study, we sequenced and characterized melanoma differentiation-associated antigen 5 (LcMDA5), laboratory of genetics and physiology 2 (LcLGP2) and mitochondrial antiviral signaling protein (LcMAVS) from large yellow croaker (Larimichthys crocea). The LcMDA5 encodes 969 amino acids and contains two caspase-associated and recruitment domains (CARDs), a DExDc (DExD/H box-containing domain), a HELICc (helicase superfamily C-terminal domain) and a C-terminal regulatory domain (RD). The LcLGP2 encodes 679 amino acids and contains a DExDc, a HELICc and a RD. The LcMAVS encodes 512 amino acids and contains a CARD, a proline-rich domain, a transmembrane helix domain and a putative TRAF2-binding motif ((269)PVQDT(273)). Phylogenetic analyses showed that all the three genes of large yellow croaker are clustered together with their counterparts from other teleost fishes. The Real-time PCR analyses showed that all the three genes were found to be constitutively expressed in all examined tissues in large yellow croaker, but all with relatively low expression levels. Expression analyses showed that the three genes were all rapidly and significantly upregulated invivo after poly (I:C) challenge in peripheral blood, liver, spleen and head kidney tissues. The results indicate that the LcMDA5, LcLGP2 and LcMAVS might play important roles in antiviral immune responses.

Transcriptome analysis demonstrate widespread differential expression of long noncoding RNAs involve in Larimichthys crocea immune response.

Long noncoding RNAs (lncRNAs) are a class of transcripts that longer than 200bp and do not encode proteins. Recent genome-wide studies of vertebrate transcriptomes have annotated lncRNAs that are expressed in various tissues and development stages. The draft genome and several transcriptome sequencing data sets have been collected for the study of protein-coding genes in large yellow croaker (Larimichthys crocea), but little is known about the expression and functional roles of lncRNAs in this species. In order to obtain a catalog of lncRNAs for large yellow croaker, several RNA-seq datasets were integrated from various tissues including egg, muscle, liver, and spleen. A total of 48,953 high-confidence transcripts were reconstructed in 38,017 loci, recovering the most of expressed reference transcripts while thousands of novel expressed loci have been identified. The tissue expression profile revealed that most lncRNAs were specifically enriched in different tissues. A stringent set of 210 lncRNAs were identified as being specifically expressed in spleen and potentially involved in immune response. Our study first systematically identify lncRNAs in large yellow croaker, benefiting the future genomic study of this species.

Effects of dietary size-fractionated fish hydrolysates on growth, activities of digestive enzymes and aminotransferases and expression of some protein metabolism related genes in large yellow croaker (Larimichthys crocea) larvae

The present study was conducted to investigate the effects of size-fractionated fish hydrolysates on growth, activities of digestive enzymes and aminotransferases and expression of some protein metabolism related genes in large yellow croaker (Larimichthys crocea) larvae. Fish (initial body weight 3.15 ± 0.15 mg) were fed for 30 days with four diets: the control diet was produced using fish meal (FM) as the main protein source and other three diets were formulated with permeate after ultra-filtration of fish hydrolysate (PUFH), retentate after ultra-filtration of fish hydrolysate (RUFH) or non-ultrafiltered fish hydrolysate (NUFH) replacing approximately 40% FM. The results showed that specific growth rate and survival rate were significantly lower in fish fed diets with NUFH and RUFH than that of fish fed the control diet (P < 0.05) and there were no significant differences between PUFH-40 group and the control group (P > 0.05). Specific activities of digestive enzymes and the ratio “pancreatic enzyme in intestinal segment/pancreatic enzyme in pancreatic segment” were not significantly different between fish fed diets with PUFH and fish meal and they were both higher than that of fish fed diets with NUFH and RUFH (P < 0.05). Specific activities of both alanine and aspartate aminotransferases were significantly higher in fish fed the diet with PUFH than that of fish fed other diets (P < 0.05). Transcription of peptide transporter 1 (PepT1), cholecystokinin (CCK) and trypsin was significantly down-regulated in fish fed diets with NUFH and RUFH (P < 0.05). No significant differences were observed in the target of rapamycin (TOR) gene expression of fish among dietary treatments (P > 0.05). These results suggest that when replacing 40% FM, PUFH seems optimal for large yellow croaker larvae compared to NUFH and RUFH and size-fractionated fish hydrolysates could significantly affect digestion and absorption of protein in large yellow croaker larvae by altering the mRNA expression levels of CCK, PepT1 and trypsin.

Molecular characterization and functional analysis of two distinct liver-expressed antimicrobial peptide 2 (LEAP-2) genes in large yellow croaker (Larimichthys crocea)

Liver-expressed antimicrobial peptide 2 (LEAP-2) plays a vital role in the host innate immune system. In the present study, two LEAP-2 genes (LcLEAP-2A and LcLEAP-2C) from large yellow croaker (Larimichthys crocea) were cloned, both of which consist of 3 exons and 2 introns. The LcLEAP-2A transcripts were expressed in a wide range of tissues, with the highest mRNA levels found in the liver and intestine, while LcLEAP-2C transcripts showed obvious lower mRNA levels in all tested tissues compared to LcLEAP-2A. Upon infection by Vibrio alginolyticus, LcLEAP-2A transcripts were significantly up-regulated in liver, trunk kidney, spleen, head kidney, and gill, but down-regulated in intestine. In addition, significant up-regulation of LcLEAP-2C transcripts were also detected in all tissues tested, including intestine. The LcLEAP-2A and LcLEAP-2C mature peptides were chemically synthesized and found to exhibit selective antimicrobial activity in vitro against various species of bacteria. LcLEAP-2C, but not LcLEAP-2A, had antimicrobial activity against V. alginolyticus. Moreover, LcLEAP-2C treatment at low concentrations was evaluated and found to improve survival rate in V. alginolyticus-infected large yellow croaker, resulting in a decrease in bacterial load and expression of inflammatory cytokines. These results suggest that LcLEAP-2 isoforms play an important role in innate immunity by killing bacteria and inhibiting early inflammatory response in large yellow croaker.

Family structure and phylogenetic analysis of odorant receptor genes in the large yellow croaker (Larimichthys crocea)

BackgroundChemosensory receptors, which are all G-protein-coupled receptors (GPCRs), come in four types: odorant receptors (ORs), vomeronasal receptors, trace-amine associated receptors and formyl peptide receptor-like proteins. The ORs are the most important receptors for detecting a wide range of environmental chemicals in daily life. Most fish OR genes have been identified from genome databases following the completion of the genome sequencing projects of many fishes. However, it remains unclear whether these OR genes from the genome databases are actually expressed in the fish olfactory epithelium. Thus, it is necessary to clone the OR mRNAs directly from the olfactory epithelium and to examine their expression status.ResultsEighty-nine full-length and 22 partial OR cDNA sequences were isolated from the olfactory epithelium of the large yellow croaker, Larimichthys crocea. Bayesian phylogenetic analysis classified the vertebrate OR genes into two types, with several clades within each type, and showed that the L. crocea OR genes of each type are more closely related to those of fugu, pufferfish and stickleback than they are to those of medaka, zebrafish and frog. The reconciled tree showed 178 duplications and 129 losses. The evolutionary relationships among OR genes in these fishes accords with their evolutionary history. The fish OR genes have experienced functional divergence, and the different clades of OR genes have evolved different functions. The result of real-time PCR shows that different clades of ORs have distinct expression levels.ConclusionWe have shown about 100 OR genes to be expressed in the olfactory epithelial tissues of L. crocea. The OR genes of modern fishes duplicated from their common ancestor, and were expanded over evolutionary time. The OR genes of L. crocea are closely related to those of fugu, pufferfish and stickleback, which is consistent with its evolutionary position. The different expression levels of OR genes of large yellow croaker may suggest varying roles of ORs in olfactory function.

Cloning and molecular characterization of lycC1INH genes in large yellow croaker ( Pseudosciaena crocea)

Complement component 1 inhibitor (C1INH) is a crucial protein in controlling activation of many plasma mediator pathways and can directly interact with Gram negative bacteria. The full-length cDNA of lycC1INH gene was identified from the large yellow croaker. It is of 2046 nucleotides (nt) encoding a protein of 599 amino acids, with a 5′-untranslated region of 99 nt and a 3′-untranslated region of 147 nt including the poly (A) tail. The deduced protein contains a C-terminal serpin (serine protease inhibitor) domain, and two N-terminus immunoglobulin domains without significant homology to other species. Western blot analysis of the protein expression showed that the expression of lycC1INH was obviously up-regulated in liver, spleen and head kidney of the fish challenged by attenuated live Vibrio anguillarum strain. This indicated that lycC1INH might be involved in the immune response of large yellow croaker to bacterial challenge.

Cloning and molecular characterization of two complement Bf/C2 genes in large yellow croaker ( Pseudosciaena crocea)

Complement components factor B and C2 are two crucial proteases in the alternative pathway (AP) and classical pathway (CP). Two Bf/C2 cDNAs, LycBf/C2A and LycBf/C2B were isolated from the large yellow croaker ( Pseudosciaena crocea) by suppression subtractive hybridization (SSH) and rapid amplification of cDNA ends (RACE). Through sequence alignment and computer 3D modeling analysis, we found that both of the deduced proteins contain three complement control protein (CCP) modules, a von Willebrand factor A (vWFA) domain, and one serine protease (SP) domain. Both structural analysis and phylogenetic analyses suggested that LycBf/C2A is more like human factor B than human C2 while LycBf/C2B is more human C2-like. After that, RT-PCR assay showed that LycBf/C2A and LycBf/C2B were mostly expressed in liver, albeit detectable in other tissues. Finally, after being infected with attenuated live Vibrio anguillarum strain, the expression level of LycBf/C2A and LycBf/C2B were found remarkably up-regulated in liver, spleen and kidney, indicating that the two complement factors play a pivotal role in the immune response to bacterial challenge in large yellow croaker.

Molecular characterization and expression analysis of MHC class II α and β genes in large yellow croaker (Pseudosciaena crocea)

MHC class II molecules play an important role in the activation of CD4(+) T cells, which are the central orchestrating cells of an immune response. Here, we report the cloning of MHC class II alpha and beta cDNAs from large yellow croaker (Pscr-DAAs and Pscr-DAB) by expressed sequence tags analysis and RACE-PCR techniques. Three different class II alpha and two class II beta sequences were obtained from spleens of two individual fish. Each of the three class II alpha sequences encodes a polypeptide of 239 amino acids while the two class II beta cDNA sequences encode for a protein of 249 aa. All the characteristic features of MHC class II chain structure could be identified in the deduced proteins of three class II alpha and two class II beta sequences, including the leader peptide, alpha1/beta1 and alpha2/beta2 domains, connecting peptide and transmembrane and cytoplasmic regions, as well as conserved cysteines and N-glycosylation site. RT-PCR analysis showed that MHC class II alpha and beta mRNAs were broadly expressed in various tissues examined, although at different levels. Upon stimulation with inactivated trivalent bacterial vaccine or polyinosinic polycytidylic acid (poly(I:C)), the expression levels of both alpha and beta genes were obviously up-regulated in intestine, kidney and spleen. Real-time PCR analysis demonstrated that the expression levels of class II alpha and beta were quickly up-regulated in spleen, kidney, and intestine at 12 h after induction with poly(I:C), while their expression levels significantly increased at 48 h upon immunization with bacterial vaccine, indicating that the up-regulation of both class II alpha and beta expression was induced by bacterial vaccine or poly(I:C) at the early phase of induction, and that class II alpha and beta transcripts were quicker up-regulated by poly I:C than by bacterial vaccine.