Lin Haoran

Polygenic expression of somatostatin in orange-spotted grouper (Epinephelus coioides): Molecular cloning and distribution of the mRNAs encoding three somatostatin precursors

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.

Transcriptome Analysis of the Trachinotus ovatus: Identification of Reproduction, Growth and Immune-Related Genes and Microsatellite Markers

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.

Production of Recombinant Orange-Spotted Grouper (Epinephelus coioides) Luteinizing Hormone in Insect Cells by the Baculovirus Expression System and Its Biological Effect

Abstract 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.

Comparative analyses of sequence structure, evolution, and expression of four somatostatin receptors in orange-spotted grouper (Epinephelus coioides).

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.

Differences in mGnRH and cGnRH-II contents in pituitaries and discrete brain areas of Rana rugulosa W. according to age and stage of maturity

(1) In tadpoles, chicken-II gonadotropin-releasing hormone (cGnRH-II) could be measured in the brains before metamorphosis, but mammalian gonadotropin-releasing hormone (mGnRH) did not appear until the stage of metamorphosis, i.e. cGnRH-II appeared earlier than mGnRH during ontogenesis. (2) During the metamorphic climax, mGnRH content increased more rapidly than cGnRH-II; the content of mGnRH was about two times of that of cGnRH-II. (3) In juveniles and adults, the content of mGnRH and cGnRH-II, and the distribution pattern of mGnRH (but not cGnRH-II) in the brains and pituitaries changed with age and stages of gonadal development. mGnRH mainly distributed in the rostral brain areas, whereas cGnRH-II had a widespread distribution in the brain. (4) Both mGnRH and cGnRH-II were present in the pituitaries at each stage of maturity. The gonadotropin-releasing hormone (GnRH) content at sexually maturity was significantly higher than that at other stages of gonadal development, and the content of mGnRH was about 15-18 times of that of cGnRH-II. (5) These results suggest that both mGnRH and cGnRH-II are potentially involved in the direct regulation of pituitary gonadotropes, and mGnRH may be the major active form, cGnRH-II may also serve as a neurotransmitter or neuromodulator in the brain.

Construction of brain cDNA libraries and molecular cloning and expression analysis of GnRH gene in Marbled eel (Anguilla marmorata).

Marbled eel(Anguilla marmorata) is the secondary protected animal in China.In order to save the genetic information of this rarity and clone the function genes on growth,development and reproduction,a cDNA library was constructed by CloneminerTM kit from brain of marbled eel.The titer of the amplified cDNA library was 4.3×106 cfu/mL,the total of recombinants was 5.16×107 cfu,and the percentage of recombinant efficiency was about 99.6%,the exogenous inserts of the recombinants was from 0.43kb to 3.2kb and the average size was 1532 bp.These results showed that cDNA library had excellent quality.Two types of GnRH(mGnRH and cGnRH-II) cDNA sequences were isolated from cDNA library by PCR.Sequence analysis showed that mGnRH cDNA contained an open reading frame(ORF) of 276 bp and encoded 91 amino acid residues,which consisting of a 22-amino acid signal peptide precursor(1—22 amino acid residues),mGnRH decapeptide(23—32 amino acid residues),3-amino acid signal processing site(33—35 amino acid residues),and a 56-amino acid GnRH-associated peptide(36—91 amino acid residues).cGnRH-II cDNA open reading frame(ORF) contained 264 bases encoded 87 amino acid residues,which consisting of a 24-amino acid signal peptide precursor(1—24 amino acid residues),cGnRH-II decapeptide(25—34 amino acid residues),3-amino acid signal processing site(34—36 amino acid residues),and a 50-amino acid GnRH-associated peptide(37—87 amino acid residues).The homology analysis showed that the percentage of mGnRH and cGnRH-II precursor sequence identity with Japanese eel Anguilla japonica is 98%,with fishes of Salmoniformes,Perciformes and Pleuronectiformes is 73%—78%.However,it was relative low with fishes of Cypriniformes(63%—67%).Phylogenetic tree analysis ranked the fish GnRH as three distinct groups,mGnRH and sbGnRH group,cGnRH-II group and sGnRH group,respectively.Expression analysis by RT-PCR showed that cGnRH-II and mGnRH gene expression had no obvious differences between female and male marbled eel individuals,but mGnRH gene could be expressed in more tissues than cGnRH-II.mGnRH were detected in forebrain,midbrain,hindbrain,pituitary,hypothalamus,liver,heart,spleen,kidney,intestine,gill,ovary and testis,while expression of cGnRH-II was mainly limited to forebrain,midbrain,hindbrain,ovary and testis.The present work provided evidence of two GnRH in Marbled eel reproductive system and suggested an important role of mGnRH in reproduction.