Expression and purification of ecdysteroid-regulated protein from Chinese mitten crab Eriocheir sinensis in E. coli.

The glutathione S-transferase (GST) fusion protein system is widely used for high-level expression and efficient purification of recombinant proteins from bacteria. The goal of this study was to clone, efficiently express and purify the ecdysteroid-regulated protein (ERP) in the form of a GST fusion protein. The mature peptide-coding cDNA fragment was extracted from Chinese mitten crap (Eriocheir sinensis), and then after using PCR to obtain the open reading frame, a recombinant plasmid designated pGEX-4T-1_ERP was successfully generated and showed to efficiently express the ERP fusion protein as determined by SDS-PAGE. The resulting expressed protein was successfully purified by a combination of affinity and conventional chromatographic methods. After purification, the recombinant protein showed the expected size of 41 kDa on SDS-PAGE gels which was further confirmed by mass spectrometry and western blotting. Purification of recombinant protein was achieved by fast protein liquid chromatography. About 2.4 mg/l recombinant protein with purity more than 80 % was obtained.

Cloning, promoter analysis and expression of the tumor necrosis factor receptor-associated factor 6 (TRAF6) in Japanese scallop (Mizuhopecten yessoensis).

Tumor necrosis factor receptor-associated factor 6 (TRAF6) is a key adaptor molecule for the tumor necrosis factor superfamily and Toll-like/interleukin-1 receptor superfamily. It plays an important role in innate and adaptive immunity. The TRAF6 of Japanese scallop Mizuhopecten yessoensis (designated as MyTRAF6) was identified and characterized in this study. The full-length cDNA of MyTRAF6 was 2,407 bp, which consisted of 239-bp 5'-terminal untranslated region, 1,974-bp open reading frame encoding a polypeptide of 657 amino acids, 194-bp of 3'-terminal untranslated region followed by a canonical polyadenylation signal sequence AATAAA and a poly (A) tail. The predicted amino acid sequence of MyTRAF6 contained the characteristic motifs of TRAF proteins, including a Zinc finger of RING-type, two Zinc fingers of TRAF-type, and a MATH (meprin and TRAF homology) domain. It had an overall identity of 43-96% with those of other TRAF6s, the highest identity (96%) with Chlamys farreri TRAF6, and the least identity (43%) with Meleagris gallopavo TRAF6. Phylogenetic analysis classified MyTRAF6 as a true TRAF6 ortholog. In addition, the promoter of MyTRAF6 was also identified by genome walking. It contained several potential transcription factor-binding sites and three single nucleotide polymorphisms. qRT-PCR analysis revealed that MyTRAF6 was highly expressed in hemocytes of adult M. yessoensis. MyTRAF6 transcript level in the hemocytes reached a maximum 6 h after Vibrio anguilarum challenge. The results indicated that MyTRAF6 may fulfill an important function during M. yessoensis bacterial infection. It could be a key effector molecule involved in the innate defense of molluscs.

Identification and expression of a putative LPS-induced TNF-α factor from Asiatic hard clam Meretrix meretrix.

LPS-induced TNF-α (LITAF) is a novel transcriptional factor that mediates the expression of inflammatory cytokines in LPS-induced processes. In the present study, the full-length cDNA encoding LITAF (designated as Mm-LITAF) was identified from Asiatic hard clam, Meretrix meretrix, by expressed sequence tag and rapid amplification of cDNA ends (RACE) approaches. The full-length cDNA of Mm-LITAF was 1653 bp, consisting of a 5' untranslated region (UTR) of 91 bp, a 3'UTR of 1166 bp with one cytokine RNA instability motif (ATTTA) and one polyadenylation signal (AATAAA), and an open reading frame (ORF) of 396 bp encoding a polypeptide of 131 amino acids with a theoretical isoelectric point of 7.49, and predicted molecular weight of 14.47 kDa. The deduced amino acid of Mm-LITAF shared 29-63% similarity with the LITAFs from other species, indicating that Mm-LITAF should be a member of the LITAF family. Two highly conserved CXXC motifs forming a compact Zn(2+)-binding structure were also identified in Mm-LITAF. A quantitative reverse transcriptase real-time PCR (qRT-PCR) assay was developed to assess the expression of Mm-LITAF mRNA in different tissues, and the temporal expression of Mm-LITAF in clams challenged with Vibrio anguillarum. The mRNA transcript of Mm-LITAF could be detected in all the examined tissues with the highest expression level in the gill. Mm-LITAF expression was up-regulated significantly at 16 h in the gill and at 8 h in haemocytes after bacterial challenge, respectively. These results suggest that the Mm-LITAF is a constitutive and inducible acute-phase protein that perhaps involved in the innate immune response of hard clam.

Intracellular Cu/Zn superoxide dismutase (Cu/Zn-SOD) from hard clam Meretrix meretrix: its cDNA cloning, mRNA expression and enzyme activity.

Hard clam (Meretrix meretrix) is an economically important bivalve in China. In the present study, a gene coding for an intracellular Cu/Zn-SOD was cloned and characterized from hard clam. The full-length cDNA of this Cu/Zn-SOD (designated as Mm-icCuZn-SOD) consisted of 1,383 bp, with a 462-bp of open reading frame (ORF) encoding 153 amino acids. Several highly conserved motifs, including the Cu/Zn binding sites [H(46), H(48), H(63), and H(119) for Cu binding; H(63), H(71), H(80), and D(83) for Zn binding], an intracellular disulfide bond and two Cu/Zn-SOD signatures were identified in Mm-icCu/Zn-SOD. The deduced amino acid sequence of Mm-icCu/Zn-SOD has a high degree of homology with the Cu/Zn-dependent SODs from other species, indicating that Mm-icCu/Zn-SOD should be a member of the intracellular Cu/Zn-dependent SOD family. Real-time PCR analysis showed that the highest level of Mm-icCu/Zn-SOD expression was in the hepatopancreas, while the lowest level occurred in the hemocytes. Hard clam challenged with Vibrio anguillarum showed a time-dependent increase in Mm-icCu/Zn-SOD expression that reached a maximum level after 6 h. Mm-icCu/Zn-SOD purified as a recombinant protein expressed in E. coli retained a high level of biological activity, 83 % after 10 min incubation at 10-50 °C, and more than 87 % after incubation in buffers with pH values between 2.2 and 10.2. These results indicated that Mm-icCu/Zn-SOD may play an important role in the innate immune system of hard clam.

The innate immune-related genes in catfish.

Catfish is one of the most important aquaculture species in America (as well as in Asia and Africa). In recent years, the production of catfish has suffered massive financial losses due to pathogen spread and breakouts. Innate immunity plays a crucial role in increasing resistance to pathogenic organisms and has generated increasing interest in the past few years. This review summarizes the current understanding of innate immune-related genes in catfish, including pattern recognition receptors, antimicrobial peptides, complements, lectins, cytokines, transferrin and gene expression profiling using microarrays and next generation sequencing technologies. This review will benefit the understanding of innate immune system in catfish and further efforts in studying the innate immune-related genes in fish.

[Cloning and expression analysis of lipopolysaccharide-induced TNF-a(LITAF) of Japanese scallop (Mizuhopecten yessoensis)].

The lipopolysaccharide-induced TNF-alpha factor (LITAF) is an inflammatory cytokine, which plays an important role in innate immunity system. Based on the expressed sequence tag (EST) of Japanese scallop (Mizuhopecten yessoensis), the cDNA of LITAF gene was amplified using rapid amplification of cDNA ends (RACE) approach. Results showed that the full-length cDNA of LITAF is 1 551 bp consisting of a 5' untranslated region (UTR) of 76 bp, a 3' UTR of 1 001 bp, and an open reading frame (ORF) of 474 bp encoding a polypeptide of 157 amino acids, and there is a conserved LITAF domain in amino acid sequences. The estimated molecular mass is 16.99 kDa and the theoretical isoelectric point is 6.24. The total length of LITAF is 3 698 bp, which includes three exons and two introns. Real-time quantitative PCR was carried out to measure LITAF mRNA expression in adult tissues and monitor mRNA expression patterns during embryonic development after bacteria (Vibrio anguillarum) challenged. The expression level of LITAF mRNA was detected in all the adult tissues with the highest in the kidneys. The trochophore owns the highest expression level of LITAF in embryonic development. LITAF expression showed significant difference(P<0.01)between the control and bacteria challenged specimens at 36 h. These results suggest that the LITAF should be a member of the LITAF family that perhaps involved in the innate immune response of Japanese scallop.

Identification of host-defense genes and development of microsatellite markers from ESTs of hard clam Meretrix meretrix.

The hard clam Meretrix meretrix is an economically important shellfish in China. However, genomic research on this species is still at early stage, and few genomic resources are available. The objective of the present study was to generate expressed sequence tags (ESTs), and identify host-defense genes and microsatellite markers for M. meretrix. Three cDNA libraries for intestine, mantle and hepatopancreas were constructed using highly efficient SMART (Switching Mechanism At 5' end of the RNA Transcript) method. A total of 3224 random clones were single-pass sequenced from 5'-ends, resulting in 3129 high-quality (>100 bp) ESTs averaging 734 bp. All the ESTs were assembled by software Cap 3, producing 1796 unigenes-1490 singletons and 306 contigs. All the unigenes were compared to the public protein database using tblastx, and 696 (38.8%) were homologues to known genes while the remaining 1100 (61.2%) appeared to be novel sequences. A total of 31 EST clusters were related to immune and defense functions. They included immune recognition receptors, proteases and protease inhibitors, and other immune-related genes. The screening of 1796 unigenes identified 55 (3.1%) microsatellite-containing sequences, with 20 having sufficient flanking sequences for primer design. Polymerase chain reaction amplification was successful for 12 primer pairs and 7 of them showed polymorphic. The EST collection and microsatellite markers obtained in this study provide a useful resource for further gene discovery and population genetic analysis in M. meretrix.

The complete mitochondrial genome of the hard clam Meretrix meretrix.

Veneridae is a diverse, commercially important, and cosmopolitan family. Here we present the complete mitochondrial genome of the hard clam Meretrix meretrix (Bivalvia: Veneridae). The entire mitochondrial genome (mitogenome) sequence of M. meretrix is 19,826 bp in length, and contains 37 genes including 12 protein-coding genes, 2 ribosomal RNAs, and 23 tRNAs. All genes are encoded on the heavy strand. In contrast to the typical animal mitochondrial genome, it lacks the protein-coding gene ATP8, and has only one copy of the tRNA(Ser) gene, but three duplications of the tRNA(Gln), which is the first report among the present molluscan mtDNAs. We observed that the gene arrangement between M. meretrix and M. petechialis is same except one more tRNAGln gene in M. meretrix., and the sequence similarity is as high as 99%, indicating that M. petechialis and M. meretrix could be treated as a junior synonym of M. meretrix. Maximum Likelihood and Bayeslan analysis of 12 concatenated protein-coding amino acid sequences place the Unionidae as a sister group to other bivalves, which reflects the general opinion that the Unionidae deverged very early in Bivalvia evolution.

Major histocompatibility complex class IIA and IIB genes of the spotted halibut Verasper variegatus: genomic structure, molecular polymorphism, and expression analysis.

The major histocompatibility complex (MHC) is a large genomic region characterized by extremely high polymorphism and its association with resistance/susceptibility to disease in vertebrates. In this study, the full lengths of MHC IIA and IIB cDNA were obtained from spotted halibut (Verasper variegates) by expressed sequence tag (EST) and rapid amplification of cDNA ends (RACE) approaches. The genomic structure, molecular polymorphism, and expression patterns were examined to study MHC II gene functions in fish. As in other teleosts, the genomic structure of the spotted halibut MHC IIA contained 4 exons and 3 introns. The deduced amino acid sequence of the class IIA molecule shared 28-79% similarity with those of teleosts and mammals. Nine class IIA alleles were identified from five individuals. Three alleles originating from a single individual suggested the existence of at least two class IIA loci in the genome. Six exons and 5 introns were identified from spotted halibut MHC IIB, and the deduced amino acid sequence shared 33-79% similarity with those of teleosts and mammals. Twelve alleles were identified, among which five were observed in a single individual, which suggested at least three class IIB loci. Quantitative real-time PCR analysis revealed the presence of class IIA and IIB transcripts in nine normal tissues with high expression level in kidney and gill. Furthermore, MHC IIA and IIB are probably two candidates of immune molecules involved in the acute-phase response in spotted halibut, because their transcriptional levels were significantly up-regulated in blood and liver after bacterial challenge.

Construction of genetic linkage maps and comparative genome analysis of catfish using gene-associated markers.

A genetic linkage map of the channel catfish genome (N=29) was constructed using EST-based microsatellite and single nucleotide polymorphism (SNP) markers in an interspecific reference family. A total of 413 microsatellites and 125 SNP markers were polymorphic in the reference family. Linkage analysis using JoinMap 4.0 allowed mapping of 331 markers (259 microsatellites and 72 SNPs) to 29 linkage groups. Each linkage group contained 3-18 markers. The largest linkage group contained 18 markers and spanned 131.2 cM, while the smallest linkage group contained 14 markers and spanned only 7.9 cM. The linkage map covered a genetic distance of 1811 cM with an average marker interval of 6.0 cM. Sex-specific maps were also constructed; the recombination rate for females was 1.6 times higher than that for males. Putative conserved syntenies between catfish and zebrafish, medaka, and Tetraodon were established, but the overall levels of genome rearrangements were high among the teleost genomes. This study represents a first-generation linkage map constructed by using EST-derived microsatellites and SNPs, laying a framework for large-scale comparative genome analysis in catfish. The conserved syntenies identified here between the catfish and the three model fish species should facilitate structural genome analysis and evolutionary studies, but more importantly should facilitate functional inference of catfish genes. Given that determination of gene functions is difficult in nonmodel species such as catfish, functional genome analysis will have to rely heavily on the establishment of orthologies from model species.

Genetic differentiation between natural and hatchery stocks of Japanese scallop (Mizuhopecten yessoensis) as revealed by AFLP analysis.

Japanese scallop (Mizuhopecten yessoensis) is a cold-tolerant bivalve that was introduced to China for aquaculture in 1982. In this study, amplified fragment length polymorphism (AFLP) markers were used to investigate levels of genetic diversity within M. yessoensis cultured stocks and compare them with wild populations. Six pairs of primer combinations generated 368 loci among 332 individuals, in four cultured and three wild populations. High polymorphism at AFLP markers was found within both cultured and wild M. yessoensis populations. The percentage of polymorphic loci ranged from 61.04% to 72.08%, while the mean heterozygosity ranged from 0.2116 to 0.2596. Compared with wild populations, the four hatchery populations showed significant genetic changes, such as lower expected heterozygosity and percentage of polymorphic loci, and smaller frequency of private alleles, all indicative of a reduction in genetic diversity. Some genetic structures were associated with the geographical distribution of samples; with all samples from Dalian and Japan being closely related, while the population from Russia fell into a distinct clade in the phylogenetic analysis. The genetic information derived from this study indicated that intentional or accidental release of selected Japanese scallops into natural sea areas might result in disturbance of local gene pools and loss of genetic variability. We recommend monitoring the genetic variability of selected hatchery populations to enhance conservation of natural Japanese scallop resources.

Sixteen polymorphic simple sequence repeat markers from expressed sequence tags of the Chinese Mitten Crab Eriocheir sinensis.

The Chinese mitten crab (Eriocheir sinensis) is an economically important aquaculture species in China. In this study, we developed and evaluated simple sequence repeat markers from expressed sequence tags of E. sinensis. Among the 40 wild E. sinensis individuals tested, 16 loci were polymorphic. The number of alleles per locus ranged from two to ten. The observed heterozygosity ranged from 0.0667 to 0.9667, whereas the expected heterozygosity ranged from 0.0661 to 0.9051. These markers have the potential for use in genetic studies of population structure and intraspecific variation in E. sinensis.

Chromosome-level reference genome of the jellyfish Rhopilema esculentum.

BACKGROUND: Jellyfish belong to the phylum Cnidaria, which occupies an important phylogenetic location in the early-branching Metazoa lineages. The jellyfish Rhopilema esculentum is an important fishery resource in China. However, the genome resource of R. esculentum has not been reported to date. FINDINGS: In this study, we constructed a chromosome-level genome assembly of R. esculentum using Pacific Biosciences, Illumina, and Hi-C sequencing technologies. The final genome assembly was ∼275.42 Mb, with a contig N50 length of 1.13 Mb. Using Hi-C technology to identify the contacts among contigs, 260.17 Mb (94.46%) of the assembled genome were anchored onto 21 pseudochromosomes with a scaffold N50 of 12.97 Mb. We identified 17,219 protein-coding genes, with an average CDS length of 1,575 bp. The genome-wide phylogenetic analysis indicated that R. esculentum might have evolved more slowly than the other scyphozoan species used in this study. In addition, 127 toxin-like genes were identified, and 1 toxin-related "hub" was found by a genomic survey. CONCLUSIONS: We have generated a chromosome-level genome assembly of R. esculentum that could provide a valuable genomic background for studying the biology and pharmacology of jellyfish, as well as the evolutionary history of Cnidaria.

[Comparison of mitochondrial genomes of bivalves].

The structure and organization of mitochondrial genomes of 14 marine bivalves and two freshwater bivalves were analyzed using comparative genomics and bioinformatics methods. The results showed that the organization and gene order of the mitochondrial genomes of these bivalve species studied were different from each other. The size, organization, gene numbers, and gene order of mitochondrial genomes in bivalves at different taxa were different. Phylogenetic analysis using the whole mitochondrial genomes and all the coding genes showed different results-- phylogenetic analysis conducted using the whole mitochondrial genomes was consistent with the existing classification and phylogenetic analysis conducted using all coding genes not consistent with the existing classification.

[The construction of a preliminary genetic linkage map in the Japanese scallop Mizuhopecten yessoensis].

AFLP markers were used to construct the primary linkage map in a family of Mizuhopecten yessoensis. A total of 1 855 markers were generated in two parents and 52 progenies of the mapping family by using 56 AFLP primer combinations. Among the 1 855 markers, 598 were polymorphic and 354 were in agreement with the Mendelian segregating ratio of 1:1. Markers segregated according to Mendelian 1:1 ratio (P>0.05) and 23 distorted markers (0.01

Sequence and organization of the complete mitochondrial genomes of spotted halibut (Verasper variegatus) and barfin flounder (Verasper moseri).

In this work, the mitochondrial genomes for spotted halibut (Verasper variegatus) and barfin flounder (Verasper moseri) were completely sequenced. The entire mitochondrial genome sequences of the spotted halibut and barfin flounder were 17,273 and 17,588 bp in length, respectively. The organization of the two mitochondrial genomes was similar to those reported from other fish mitochondrial genomes containing 37 genes (2 rRNAs, 22 tRNAs and 13 protein-coding genes) and two non-coding regions (control region (CR) and WANCY region). In the CR, the termination associated sequence (ETAS), six central conserved block (CSB-A,B,C,D,E,F), three conserved sequence blocks (CSB1-3) and a region of 61-bp tandem repeat cluster at the end of CSB-3 were identified by similarity comparison with fishes and other vertebrates. The tandem repeat sequences show polymorphism among the different individuals of the two species. The complete mitochondrial genomes of spotted halibut and barfin flounder should be useful for evolutionary studies of flatfishes and other vertebrate species.

[MYP gene expressions at transcription level in different stages of gonad of sea urchin Strongylocentrotus intermedius and hybrids].

MYP (Major yolk protein) gene expression at transcription level in different stages of gonad of sea urchin Strongylocentrotus intermedius and hybrids (S. intermediusfemale symbolxS. nudusmale symbol) was analyzed by real-time RT-PCR. Based on normalization with 18S rRNA levels, the comparative quantities of MYP expression were determined. The expression of MYP gene in gonad showed little difference between female and male. MYP gene expression was decreased rapidly in the gonad of S. intermedius at different stages, and slowly in hybrids. The comparative quantities of MYP expression in the gonads of S. intermedius were decreased from 44.55% to 9.59% in female and from 41.17% to 1.83% in male at different stages. The comparative quantities of MYP expression in the gonads of the hybrids were decreased from 37.66% to 19.22% in female and from 36.66% to 12.55% in male at different stages. The results indicated that the difference of MYP expression was correlated with the variation caused by hybridization.

Genome-wide identification of HSP70/110 genes in sea cucumber Apostichopus japonicus and comparative analysis of their involvement in aestivation.

HSP70/110s are a subgroup of heat shock proteins and play crucial roles in protein homeostasis. HSP70/110s can enhance cell survival in response to a multitude of stressful stimuli, of which the most studied one is heat stress. To perform a systematic study of HSP70/110s in sea cucumber Apostichopus japonicus, 15 HSP70/110 genes, including 13 HSP70s and two HSP110s, were identified and characterized from the transcriptome and genome of sea cucumber. Moderate expansion and conserved structure were found by the phylogenetic and syntenic analysis. Differential expression patterns of HSP70/110s were observed in adult individuals during aestivation, with the comparison of juvenile individuals without aestivation in chronic heat stress. Tissue-specific expression profiles were found both in adult and juvenile individuals, which might indicate that the functional tissues (intestine and respiratory tree) could be restored to normal physiological activity prior to protecting and sporting tissues (body wall and muscle). Differential expression profiles were also observed between the adult and juvenile individuals, which was mainly due to the hypometabolism in aestivation. Taken together, tissue-specific pattern and individual-specific pattern were observed in the HSP70/110 expression profiles in sea cucumber during aestivation. These findings could provide early insight into the involvement of HSP70/110s in the aestivation of marine invertebrate.

Towards the ictalurid catfish transcriptome: generation and analysis of 31,215 catfish ESTs.

BACKGROUND: EST sequencing is one of the most efficient means for gene discovery and molecular marker development, and can be additionally utilized in both comparative genome analysis and evaluation of gene duplications. While much progress has been made in catfish genomics, large-scale EST resources have been lacking. The objectives of this project were to construct primary cDNA libraries, to conduct initial EST sequencing to generate catfish EST resources, and to obtain baseline information about highly expressed genes in various catfish organs to provide a guide for the production of normalized and subtracted cDNA libraries for large-scale transcriptome analysis in catfish. RESULTS: A total of 17 cDNA libraries were constructed including 12 from channel catfish (Ictalurus punctatus) and 5 from blue catfish (I. furcatus). A total of 31,215 ESTs, with average length of 778 bp, were generated including 20,451 from the channel catfish and 10,764 from blue catfish. Cluster analysis indicated that 73% of channel catfish and 67% of blue catfish ESTs were unique within the project. Over 53% and 50% of the channel catfish and blue catfish ESTs, respectively, had significant similarities to known genes. All ESTs have been deposited in GenBank. Evaluation of the catfish EST resources demonstrated their potential for molecular marker development, comparative genome analysis, and evaluation of ancient and recent gene duplications. Subtraction of abundantly expressed genes in a variety of catfish tissues, identified here, will allow the production of low-redundancy libraries for in-depth sequencing. CONCLUSION: The sequencing of 31,215 ESTs from channel catfish and blue catfish has significantly increased the EST resources in catfish. The EST resources should provide the potential for microarray development, polymorphic marker identification, mapping, and comparative genome analysis.

The catfish liver-expressed antimicrobial peptide 2 (LEAP-2) gene is expressed in a wide range of tissues and developmentally regulated.

Antimicrobial peptides (AMPs) are important components of the host's innate immune response against microbial invasion. The cysteine-rich AMPs such as defensin and hepcidin have been extensively studied, but the recently identified cysteine-rich liver-expressed antimicrobial peptide 2 (LEAP-2) has been characterized from only a few organisms. Here we cloned and sequenced the LEAP-2 cDNAs from both Channel catfish and Blue catfish. The LEAP-2 gene from Channel catfish was also sequenced and characterized. Channel catfish LEAP-2 gene consists of two introns and three exons that encode a peptide of 94 amino acids with a 28 amino acid signal peptide and a mature peptide of 41 amino acids. The amino acid sequences and gene organization were conserved between catfish and other organisms. The Channel catfish LEAP-2 gene is expressed in a wide range of tissues except brain and stomach. Its expression is developmentally regulated with no detection of mature mRNA in early stages of development. It appears that the catfish LEAP-2 gene is regulated at the level of splicing; it is constitutively transcribed, but remains unspliced until 6 days after hatching. The expression of LEAP-2 was induced in a tissue-specific manner. Its expression was upregulated in the spleen, but not in the liver and head kidney, after challenge with Edwardsiella ictaluri, the causative agent of enteric septicemia of catfish (ESC).