Cloning and Expression of NADPH-cytochrome P450 Reductase Gene in Chinese Mitten Crab, Eriocheir sinensis

Abstract NADPH-cytochromeP450 reductase (CPR) is one of the most important components of the cytochrome P450 enzyme system. In this study, a gene encoding CPR (named EsCPR) was isolated from Eriocheir sinensis using reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) methods. Analysis of the nucleotide sequence revealed a cDNA full-length of 3717 bp with an open reading frame of 2046 bp, a 5′-untranslated region of 42 bp, and a long 3′-untryganslated region of 1628bp, which encodes a protein of 681 amino acids with a predicted molecular weight of 30.7 kDa and an estimated pI of 4.82. The mature peptide shares amino acid of E. sinensis identity 82 % - 89 % to the CPR from Penaeus vannamei and Chionoecetes opilio. Tissues and developmental stage-dependent expression of EsCPR mRNA was investigated by real-time quantitative PCR. EsCPR mRNA was markedly expressed in the hepatopancreas and stomach. These results would provide valuable information for further study on the interactions between CPR and cytochrome P450 enzyme systems.

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.

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.

Transcriptome analysis of the sea cucumber (Apostichopus japonicus) with variation in individual growth.

The sea cucumber (Apostichopus japonicus) is an economically important aquaculture species in China. However, the serious individual growth variation often caused financial losses to farmers and the genetic mechanisms are poorly understood. In the present study, the extensively analysis at the transcriptome level for individual growth variation in sea cucumber was carried out. A total of 118946 unigenes were assembled from 255861 transcripts, with N50 of 1700. Of all unigenes, about 23% were identified with at least one significant match to known databases. In all four pair of comparison, 1840 genes were found to be expressed differently. Global hypometabolism was found to be occurred in the slow growing population, based on which the hypothesis was raised that growth retardation in individual growth variation of sea cucumber is one type of dormancy which is used to be against to adverse circumstances. Besides, the pathways such as ECM-receptor interaction and focal adhesion were enriched in the maintenance of cell and tissue structure and communication. Further, 76645 SSRs, 765242 SNPs and 146886 ins-dels were detected in the current study providing an extensive set of data for future studies of genetic mapping and selective breeding. In summary, these results will provides deep insight into the molecular basis of individual growth variation in marine invertebrates, and be valuable for understanding the physiological differences of growth process.

Exploring single nucleotide polymorphism (SNP), microsatellite (SSR) and differentially expressed genes in the jellyfish (Rhopilema esculentum) by transcriptome sequencing.

In this study, single nucleotide polymorphism (SNP), microsatellite (SSR) and differentially expressed genes (DEGs) in the oral parts, gonads, and umbrella parts of the jellyfish Rhopilema esculentum were analyzed by RNA-Seq technology. A total of 76.4 million raw reads and 72.1 million clean reads were generated from deep sequencing. Approximately 119,874 tentative unigenes and 149,239 transcripts were obtained. A total of 1,034,708 SNP markers were detected in the three tissues. For microsatellite mining, 5088 SSRs were identified from the unigene sequences. The most frequent repeat motifs were mononucleotide repeats, which accounted for 61.93%. Transcriptome comparison of the three tissues yielded a total of 8841 DEGs, of which 3560 were up-regulated and 5281 were down-regulated. This study represents the greatest sequencing effort carried out for a jellyfish and provides the first high-throughput transcriptomic resource for jellyfish.

The complete mitochondrial genome of Saxidomus purpuratus (Veneroida: Veneridae).

In this study, the complete mitochondrial genome of Saxidomus purpuratus is determined, which is the first complete mitochondrial genome in the genus Saxidomus. The genome was of 19 637 bp in length, including 2 rRNAs, 22 tRNAs and 12 protein-coding genes with the order of ND3 and ND5 reversed. Maximum likelihood tree based on nucleotide sequences of 12 mitochondrial PCGs was constructed, in which S. purpuratus was clustered with 3 Meretrix species. The results are expected to provide useful data for species identification and further studies of the genus Saxidomus.

Complete mitochondrial genome of Volutharpa perryi (Neogastropoda: Buccinidae).

In this study, the complete mitochondrial genome of Volutharpa perryi has been determined for the first time. The mitogenome is of 15 255 bp in length, including 13 protein-coding genes, two ribosomal RNA genes and 22 transfer RNA genes. The overall base composition is 29.6%, 38.6%, 15.6% and 16.2% for A, T, C and G, respectively. The 13 PCGs of V. perryi and other 12 mollusk species were used for phylogenetic analysis by maximum-likelihood method, and the phylogenetic tree demonstrated the close relationship of V. perryi to species of Buccinoidea. The results are expected to provide useful molecular data for species identification and further phylogenetic studies of genus Volutharpa.

The complete mitochondrial genome of Buccinum pemphigum (Neogastropoda: Buccinidae).

In this study, the complete mitogenome of Buccinum pemphigum has been determined. The complete mitochondrial genome is 15 265 bp in length, including 13 protein-coding genes, two rRNA genes and 22 tRNA genes. The total base composition is 30.1% A, 15.8% G, 15.1% C and 39.0%T, with a high AT content of 69.1%. Tree constructed using maximum-likelihood (ML) phylogenetic method, demonstrated that B. pemphigum has a close relationship to Volutharpa perryi clustered in Buccinoidea. First, this complete mitogenome of Buccinum will facilitate the development of new DNA markers for species identification.

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.

De novo assembly and characterization of spotted seal Phoca largha transcriptome using Illumina paired-end sequencing.

Spotted seal (Phoca largha) is categorized as a critically endangered species in China. The aim of this study was to investigate spotted seal transcriptome by the approach of Illumina paired-end sequencing technology. We obtained a total of 52,146,394 reads for the mixed tissues of liver and spleen from the spotted seal. The de novo assemblies yielded 354,014 contigs and 178,466 unigenes. In the transcriptome, 193 unigenes were assigned to defense mechanisms. Three unigenes encoded MHC class I and 17 unigenes encoded MHC class II. In addition, bioinformatics analysis revealed a total of 4425 simple sequence repeats (SSRs). Fifty SSRs were randomly selected to validate amplification and determine the degree of polymorphism in the genomic DNA pools. Thirty-five primer pairs successfully amplified the expected DNA fragments and detected significant polymorphism among 28 spotted seal individuals. These results would contribute to the understanding of the genetic makeup of spotted seal transcriptome and provide useful information for functional genomic research in this species.

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.

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.

Characterization of the spotted seal Phoca largha transcriptome using Illumina paired-end sequencing and development of SSR markers.

Next-generation sequencing provides a powerful new approach for developing functional genomic tools for non-model species. This study aims to investigate the spotted seal (Phoca largha) transcriptome by the approach of Illumina paired-end sequencing technology. We obtained a total of 52,146,394 reads for the mixed tissues of liver and spleen from spotted seal. The de novo assemblies yielded 354,014 contigs and 178,466 unigenes. In addition, bioinformatics analysis revealed a total of 4425 simple sequence repeats (SSRs). Fifty SSRs were randomly selected to validate amplification and determine the degree of polymorphism in the genomic DNA pools. Thirty-five primer pairs successfully amplified expected DNA fragments and detected significant polymorphism among 28 spotted seal individuals. These results contribute to the understanding of the genetic makeup of spotted seal transcriptome and provide useful information for functional genomic research in this species.

[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.

Application of taxonomic distinctness indices of littoral macroinvertebrate communities for assessing long-term variation in ecological quality status of intertidal ecosystems, northern China.

It has been increasingly recognized that taxonomic diversity indices have a number of desirable properties as an indicator for assessing ecological quality status, in particular their less sensitivity to natural habitat type and sampling effort but more to environmental stress and anthropogenic impact, and a statistical framework for the assessment of the significance of departure from expectation. Taxonomic patterns of macroinvertebrate fauna for assessing ecological quality status were studied based on six datasets collected from intertidal zones of the Yellow Sea, near Qingdao, northern China, during the period of 1989-1998. The invertebrate communities were sampled yearly at five stations with different bottom types during summer season (June). A total of 141 macroinvertebrate taxa were identified belonging 119 genera, 81 families, 34 orders, 19 classes, and 10 phyla. Multivariate analyses demonstrated that the taxonomic patterns of invertebrate fauna represented a significant variation in long-term temporal scale during the study period. The average taxonomic distinctness indices (Δ(+)) decreased to a significantly low level, while the variation in taxonomic distinctness measures (Λ(+)) increased to a significantly high level compared with the expected values from 1989 to 1998. The pairwise indices of Δ(+) and (Λ(+)) showed a decreasing and increasing trend of departure from the expected taxonomic breadth in response to the environmental stress and anthropogenic impact, respectively. These results imply that the ecological quality status has been significantly deteriorated due to the increasing environmental stress and anthropogenic impact in intertidal zones of the Yellow Sea, northern China, and that the taxonomic distinctness indices of macroinvertebrate fauna are a robust indicator for evaluating ecological quality status.

A goose-type lysozyme gene in Japanese scallop (Mizuhopecten yessoensis): cDNA cloning, mRNA expression and promoter sequence analysis.

Lysozyme is an important component of the immune response against bacteria that is characterized by its ability to break down bacterial cell-walls. We constructed a high-quality cDNA library from mantle tissue of adult Japanese scallop (Mizuhopecten yessoensis). The EST which is high homology with g-type lysozyme genes of other species was found in the cDNA library. In the present study, the complete express sequence of g-type lysozyme genes from Japanese scallop (designated as MyLysoG) was directly obtained by PCR. The complete sequence of MyLysoG cDNA consisted of a 5' untranslated region (UTR) of 25 bp, an open reading frame (ORF) of 606 bp, and a 3' UTR of 100 bp with one polyadenylation signal (AATAAA). The deduced amino acids of MyLysoG were 201 amino acids with a putative signal peptide of 18 amino acid residues. It shared the sequence similarity and the common structure features with the g-type lysozyme from other species. Quantitative reverse trancriptase real-time PCR (qRT-PCR) assay demonstrated that mRNA transcripts of g-type lysozyme could be detected in various tissues of unchallenged scallop, and the highest expression of MyLysoG was detected in hepatopancreas tissue. The temporal expression of MyLysoG in hemolymph after Vibrio anguillarum challenge was up-regulated and reached the maximum level at 3h post stimulation, and then dropped back to the original level even lower than the control group. Furthermore, a 978 bp of 5'-flanking sequence of MyLysoG was identified by genome walking, and several potential transcription factor binding sites (TFBS) were detected in the putative promoter region. One part of the MyLysoG promoter region contains nine sites of SNPs and three sites of insert-deletion (indel) polymorphisms, and these mutations were found organize into two haplotypes. The two haplotypes were associated with different TFBS. The haplotypes could be selected to analyze the transcriptional-level control of scallop g-type lysozyme gene and the scallop immune system.

Characterization of a myostatin gene (MSTN1) from spotted halibut (Verasper variegatus) and association between its promoter polymorphism and individual growth performance.

Myostatin (MSTN) is a member of the transforming growth factor-ß superfamily which could play an important role in negatively regulating skeletal muscle growth and development in mammal and non-mammal species. In the present study, a MSTN1 gene (designated as VvMSTN1) was cloned and characterized in one flatfish species, spotted halibut (Verasper variegatus). In the 3078 bp genomic sequence, three exons, two introns and a promoter sequence were identified. Sequence analysis of the promoter region revealed that it contained several cis-regulatory elements such as CAAT-box, TATA-box and E-boxes. The deduced protein sequence included a signal peptide, a TGF-ß propeptide in the N-terminal region and the TGF-ß active peptide in the C-terminal region. Phylogenetic analysis suggested that VvMSTN1 is an orthologue of teleost MSTN1 proteins which arose along with MSTN2 during a duplication event at the base of teleost evolution. Quantitative real-time PCR analysis revealed that VvMSTN1 mRNA was ubiquitously expressed in all nine tested tissues, with the most transcriptionally abundant in skeletal muscle. A primary assessment of sequence variability revealed five single nucleotide polymorphisms (SNPs) existed in the promoter region, among which three (G-653T, T-355C and G-253A) were genotyped with an advanced melting temperature (T(m))-shift method and tested for their association with growth traits (body length, body depth and total mass). Results indicated that genotype CC of locus T-355C had significantly higher growth traits than genotype TC and TT (P<0.05) in female spotted halibut. These results suggest that V. variegatus MSTN could be selected as a candidate gene for the future molecular breeding of stains with enhanced individual growth performance.

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.