ABSTRACT
BACKGROUND: The Trachinotus ovatus (Teleostei, Carangidae) is an economically important marine fish species in the world. However, the lack of genomic information regarding this species limits our understanding of the genetics and biological mechanisms in Trachinotus ovatus. In this study, high throughput transcriptome sequencing was used to obtain comprehensive genomic information in Trachinotus ovatus. PRINCIPAL FINDINGS: Transcriptome sequencing was performed by using Illumina paired-end sequencing technology. The 98,534,862 high quality reads were yielded, and were de novo assembled into 156,094 unigenes with an average sequence length of 1179 bp. Transcriptome annotation revealed that 75,586 and 67,923 unigenes were functionally annotated in the NCBI non-redundant database and Swiss-Prot protein database, respectively. Functional analysis demonstrated that 67,923 unigenes were grouped into 25 Cluster of Orthologous Groups (COG) functional categories, 37,976 unigenes were clustered into 61 Gene Ontology (GO) terms, and 38,172 unigenes were assigned to 275 different Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Based on the transcriptome dataset, a large number of unigenes associated with reproduction, growth and immunity were identified. Furthermore, a total number of 38,794 simple sequence repeats (SSRs) were discovered and 16 polymorphic loci were characterized in Trachinotus ovatus. CONCLUSION/SIGNIFICANCE: The present study is the first transcriptome analysis of a fish species belonging to the genus Trachinotus and provides a valuable genomic resource for novel gene discovery, gene expression and regulation studies, and the identification of genetic markers in Trachinotus ovatus and the other fish of the genus Trachinotus.
Subject(s)
Fishes/genetics , Immune System , Microsatellite Repeats , Reproduction/genetics , Transcriptome , Animals , Gene Expression Profiling , Polymorphism, Single NucleotideABSTRACT
Somatostatins (SSs) and somatostatin receptors (SSTRs) play important roles in the growth, development and metabolism of vertebrates. In the present study, four SSTRs were isolated from orange-spotted grouper (Epinephelus coioides), a coral fish of high commercial value cultivated in Southeast Asia. Phylogenetic tree analysis grouped the four SSTRs as two distinct groups of SSTR1 and SSTR2/3/5. Four SSTRs exhibited high homology across the vertebrates. The expression of four grouper SSTR mRNAs was studied in 11 tissues. The highest level of SSTR1 mRNA was found in forebrain. The mRNAs of SSTR2 and SSTR3 were highly expressed in pituitary, forebrain and liver. The levels of SSTR5 mRNA were low in most tissues except for pituitary and intestine. The expression of four grouper SSTR mRNAs was investigated in seven embryonic stages and five early larval development stages. The highest levels of SSTR1 and 2 mRNAs appeared during hatching, while the highest levels of SSTR3 and 5 mRNAs were found in brain vesicle stage. Intraperitoneal injection of SS14 significantly increased the levels of all four SSTR mRNAs in pituitary and SSTR1, 3 mRNAs in liver in a dose-dependent manner, but no effect on SSTR2 and 5 in liver. These observations contribute to the understanding of the evolution of SSTR family and offer information on structure, distribution and function of fish SSTRs.
Subject(s)
Biological Evolution , Fishes , Receptors, Somatostatin , Amino Acid Sequence , Animals , Base Sequence , Fishes/genetics , Fishes/metabolism , Humans , Molecular Sequence Data , Phylogeny , RNA, Messenger/genetics , RNA, Messenger/metabolism , Receptors, Somatostatin/classification , Receptors, Somatostatin/genetics , Receptors, Somatostatin/metabolism , Sequence Alignment , Sequence Analysis, DNA , Somatostatin/genetics , Somatostatin/metabolism , Tissue DistributionABSTRACT
The cDNA sequence encoding orange-spotted grouper lhb (LHbeta) and cga (GTHalpha) subunits were cocloned into baculovirus transfer vectors and expressed in insect Sf9 cells. The results showed that two bands of 15.6 kDa and 11.4 kDa could be detected by SDS-PAGE and a band of 30 kDa could be detected by native PAGE. The recombinant grouper Lh (rgLh) could stimulate the secretion of testosterone (T) and estradiol-17beta (E2) from the gonad in a static incubation system in a time-dependent, but not a dose-dependent, manner. Using in vivo bioassay, the mRNA levels of two aromatases (cyp19a1a [P450aromA] and cyp19a1b [P450aromB]), gnrh (GnRH), lhb, and cga in the pituitary, gonad, and hypothalamus were determined in different groups of orange-spotted groupers treated respectively with rgLh, human chorionic gonadotropin (hCG), and a culture medium of insect cells transformed with an expression vector without lhb and cga subunits. The mRNA levels of cyp19a1a and cyp19a1b rose dramatically after injecting rgLh intraperitoneally, which was consistent with the secretion of sex steroid hormones. Interestingly, the mRNA levels of gnrh dropped in the pituitary, hypothalamus, and gonad, and the mRNA levels of lhb and cga in the pituitary of the experimental group expressed at a higher level than that of the hCG group. These results are in accord with the long positive feedback loop of Lh on gonad sex steroid hormones and the short negative feedback loop of Lh on gnrh mRNA levels. These results indicate that the rgLh is successfully expressed by the baculovirus-insect expression system and that the rgLh has biological activity.
Subject(s)
Bass/metabolism , Luteinizing Hormone/biosynthesis , Recombinant Proteins/biosynthesis , Animals , Aromatase/genetics , Aromatase/metabolism , Baculoviridae/genetics , Bass/genetics , Cells, Cultured , Cloning, Molecular , DNA, Complementary/genetics , Genetic Vectors/genetics , Glycoprotein Hormones, alpha Subunit/biosynthesis , Glycoprotein Hormones, alpha Subunit/genetics , Gonads/drug effects , Gonads/metabolism , Humans , Insecta/cytology , Luteinizing Hormone/genetics , Luteinizing Hormone/pharmacology , Luteinizing Hormone, beta Subunit/biosynthesis , Luteinizing Hormone, beta Subunit/genetics , Plasmids/genetics , Recombinant Proteins/genetics , Recombinant Proteins/pharmacology , Testosterone/metabolism , Transfection/methodsABSTRACT
In the present study, three preprosomatostatin (PSS) cDNAs were characterized from hypothalamus of orange-spotted grouper Epinephelus coioides. The first cDNA encodes a 123-amino acid protein (PSSI) that contains the SS14 sequence at its C-terminal extremity and that is identical to that of PSSI of human and other vertebrates. The second cDNA encodes a 127-amino acid protein (PSSII) that contains the SS28 sequence with [Tyr7, Gly10]-SS14 at its C-terminus. The third cDNA encodes a 110-amino acid protein (PSSIII) that contains the somatostatin variant [Pro2]-SS14 at its C-terminal extremity. All these three PSS mRNAs were expressed in brain and pituitary with different mRNA levels. In peripheral tissues, PSSII was more widely distributed than PSSI and PSSIII. High mRNA levels of PSS were found in stomach, intestine and ovary. PSS mRNAs were detected throughout embryogeny and early larval development. Its levels increased with the embryonic development and maintained a higher level during larva developing. The mRNA distribution suggests that the three grouper PSS products play important physiological functions in adult fish as well as in cell growth and organ differentiation in embryo and larva development.
