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.

Proteomics reveals the preliminary physiological states of the spotted seal (Phoca largha) pups.

Spotted seal (Phoca largha) is a critically endangered pinniped in China and South Korea. The conventional method to protect and maintain the P. largha population is to keep them captive in artificially controlled environments. However, little is known about the physiological differences between wild and captive P. largha. To generate a preliminary protein expression profile for P. largha, whole blood from wild and captive pups were subjected to a label-free comparative proteomic analysis. According to the results, 972 proteins were identified and predicted to perform functions related to various metabolic, immune, and cellular processes. Among the identified proteins, the expression level of 51 were significantly different between wild and captive P. large pups. These differentially expressed proteins were enriched in a wide range of cellular functions, including cytoskeleton, phagocytosis, proteolysis, the regulation of gene expression, and carbohydrate metabolism. The abundances of proteins involved in phagocytosis and ubiquitin-mediated proteolysis were significantly higher in the whole blood of wild P. largha pups than in captive individuals. In addition, heat shock protein 90-beta, were determined as the key protein associated with the differences in the wild and captive P. largha pups due to the most interactions of it with various differentially expressed proteins. Moreover, wild P. largha pups could be more nutritionally stressed and have more powerful immune capacities than captive pups. This study provides the first data on the protein composition of P. largha and provides useful information on the physiological characteristics for research in this species.

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.

The complete mitochondrial genome of Chirolophis japonicus (Perciformes: Stichaeidae).

The complete mitochondrial genome of Chirolophis japonicus was sequenced for the first time in this study. It is a closed-circular molecule of 16 521 bp in length and contains 13 protein-coding genes, 2 rRNA genes, 22 tRNA genes, an origin of light strand replication (OL), and a control region (CR). The overall base composition of the heavy-strand is 25.5% A, 28.6% T, 18.3% G, and 27.6% C, and the lengths of 2 rRNA genes are 946 and 1692 bp, respectively. Maximum likelihood tree based on all the amino acid sequences of 13 mitochondrial PCGs was constructed, in which C. japonicus is close to three species of infraorder Zoarcales. These results are expected to provide useful molecular data for species identification and further phylogenetic studies of genus Chirolophis.

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 Parastichopus californicus (Aspidochirotida: Stichopodidae).

In this study, we first determined and described the complete mitogenome sequence of Parastichopus californicus, which was 16 727 bp in length. The mitochondrial genome had the canonical mitochondrial gene content, including 13 protein-coding genes, 2 rRNA genes and 22 tRNA genes. The overall base composition of the heavy-strand was 31.5% A, 18 % G, 20.6% C and 29.9% T, with a high A + T content of 61.4%. ML phylogenetic tree indicated that P. californicus and P. nigripunctatus were clustered in one branch belonging to the genus Parastichopus. This conclusion was identical to the former result by the methods of morphological taxonomy.

Complete mitochondrial genome of the marine polychaete, Marphysa sanguinea (Polychaeta, Eunicida).

The complete mitochondrial genome of Marphysa sanguinea from northern China was determined. This entire sequence was 15,159 bp in length, containing 13 protein-coding genes (PCGs), 2 rRNA genes, 22 tRNA genes and a putative control region (D-loop). The base composition of the genome was 30.7% A, 30.3% T, 26.5% C and 12.6% G. Some PCGs shared nucleotides with neighboring genes, and all 13 PCGs had ATG as a start codon. Two complete stop codons (TAA, TAG) were used by the PCGs. Compared to other marine polychaetes and oligophaetes, most of the PCGS were without any rearrangement, except for ND2 and ND3, in which the order was reversed.

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.

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.

[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.

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.

A selenium-dependent glutathione peroxidase in the Japanese scallop, Mizuhopecten yessoensis: cDNA cloning, promoter sequence analysis and mRNA expression.

Glutathione peroxidase (GPx) is an antioxidant enzyme that protects cells from oxidative damage in the innate immune responses against bacterial infections. GPx is also involved in immune defenses. In this study, we report cloning and characterization of a GPx (designated as MyGPx) coding sequences and promoter from Japanese scallop, Mizuhopecten yessoensis. The full-length 1081 nt MyGPx mRNA contained a 28 nt 5' untranslated region (UTR), a 603 nt open reading frame and a 450 nt 3' UTR containing a polyadenylation signal (AATAAA). Multiple sequence alignment revealed that amino acids essential to enzymatic function of MyGPx proteins were highly conserved. A 1628 nt 5'-flanking sequence of MyGPx was identified by genome walking. Here, several potential transcription factor binding sites were detected in the putative promoter region, and nine single nucleotide polymorphisms (SNPs) were found in the 5' sequence flanking the promoter region. Quantitative Real time PCR (qRT-PCR) was employed to measure GPx mRNA expression in adult tissues and monitor mRNA expression patterns during embryonic development and following stimulation by the bacteria Vibrillo anguillarum. Collectively, the results suggest that MyGPx fulfills an important function during M. yessoensis development and may be an important immune effector in adult molluscs.

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.

[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