| We take cultured Oplegnathus fasciatus as the experiment fish and researchthe physiological and endocrine characteristics. The research mainlyfocused on the full-length cDNA cloning，tissue expression and expressioncharacteristics of different ovarian development stage of GonadotropinHormone and Membrane progestin receptorand the regular pattern of sexsteroid hormones in different ovarian development stage using histology,homology cloning, rapid amplification of cDNA ends andradioimmunoassay methods. The research would be beneficial to theregulation of artificial breeding in Oplegnathus fasciatus, and also providesa certain value to the reproductive regulation of Oplegnathus fasciatus.1. Molecularcloning and the expression characteristics of gonadotropinhormone alpha subunit in Oplegnathus fasciatusA full length cDNA sequence of gonadotropin hormone common alphasubunit was cloned from the pituitary of Oplegnathus fasciatus usinghomology cloning and RACE techniques. The GtHα cDNA sequenceconsisted of897base pairs nucleotides, including249base pairs at5’untranslation region,249base pairs at3’ untranslation region and an openreading frame with399base pairs nucleotides. This cDNA sequence encoded a precursor protein of132amino acids and the former38aminoacids residues formed a signal peptide. The putative peptide contained10cysteine residues and2N-glycosylation sites residues, in the middle of122and124cysteine were separated by a histidine residue and consequentlyformed a CXC chemokine characteristic structure. In addition, the3’containing a polyadenylation signal ATTAAA. Its predicted molecularweight was14.59KDa and the oelectric point was8.41.Comparison of theamino acid sequences and phylogentic analysis showed that theOplegnathus fasciatusGtHα had the close relationship with Perciformes，which was highest similarity with Dicentrarchus labrax(98%).The results displayed that the level of GtHα mRNA was higher in pituitary,ovary, brain, and trace expression in heart, liver, stomach, intestine, muscle,pyloric caecum and was not expressed in kidney, Head kidney, spleen, gillby Real-time Quantitative PCR technique, it was highest in pituitary. Thereproductive cycle showed that the level of GtHα mRNA in pituitary wasmaximum in stageⅤof ovarian development and the level of GtHα mRNAin ovary was maximum in stage Ⅳ of ovarian development, however, thelevel of GtHα mRNA in brain was lowest in stage Ⅴ of ovariandevelopment.2. Molecular cloning and the expression characteristics offollicle-stimulating hormone in Oplegnathus fasciatus A full length cDNA sequence of follicle-stimulating hormone was clonedfrom the pituitary of Oplegnathus fasciatus using homology cloning andRACE techniques. The FSHβ cDNA sequence consisted of554base pairsnucleotides, including an open reading frame with363base pairsnucleotides. This cDNA sequence encoded a precursor protein of120amino acids and the former18amino acids residues formed a signalpeptide. The putative peptide contained12cysteine residues and1N-glycosylation sites residues, in the middle of255and257cysteine wereseparated by a glutamicacid residue and consequently formed a CXCchemokine characteristic structure. In addition, the3’ containing apolyadenylation signal AAATAA. Its predicted molecular weight was12.96KDa and the oelectric point was6.05. Comparison of the amino acidsequences and phylogentic analysis showed that the Oplegnathus fasciatusFSHβ had the close relationship with Perciformes，which was highestsimilarity with Lateolabrax japonicus (88%).The results displayed that the level of FSHβ mRNA was higher inpituitary, ovary, and trace expression in brain, heart, liver, stomach,intestine, spleen, gill, muscle, pyloric caecum and was not expressed inkidney and Head kidney by Real-time Quantitative PCR technique, it washighest in pituitary. The reproductive cycle showed that the level of FSHβmRNA in pituitary was maximum in stage Ⅳ of ovarian development,however, the level of FSHβ mRNA in ovary and brain wasminimum in stageⅤof ovarian development.3．Molecular cloning and the expression characteristics ofLuteinizingHormone in Oplegnathus fasciatusA full length cDNA sequence of follicle-stimulating hormone was clonedfrom the pituitary of Oplegnathus fasciatus using homology cloning andRACE techniques. The LHβ cDNA sequence consisted of857base pairsnucleotides, including an open reading frame with450base pairsnucleotides. This cDNA sequence encoded a precursor protein of149amino acids and the former24amino acids residues formed a signalpeptide. The putative peptide contained12cysteine residues and1N-glycosylation sites residues, In the middle of4and5cysteine were thespecific characteristic structure Cys-Ser-Gly-His (CSGH) of Teleost. Inaddition, the3’containing a polyadenylation signal AATAAA. Its predictedmolecular weight was16.56KDa and the oelectric point was5.52.Comparison of the amino acid sequences and phylogentic analysis showedthat the LHβ had the close relationship with Perciformes，which washighest similarity with Epinephelus coioides (98%).The results displayed that the level of LHβ mRNA was higher in pituitary,ovary, and trace expression in brain, heart, liver, stomach, intestine, spleen,gill, muscle, pyloric caecum, kidney and was not expressed in Head kidney by Real-time Quantitative PCR technique, it was highest in pituitary. Thereproductive cycle showed that the level of LHβ mRNA in pituitary andovary was maximum in stageⅤof ovarian development,however, the levelof LHβ mRNA in brain was minimum in stageⅤof ovarian development.4．Molecular cloning and the expression characteristics of Membraneprogestin receptor in Oplegnathus fasciatusA full length cDNA sequence of follicle-stimulating hormone was clonedfrom the ovary of Oplegnathus fasciatus using homology cloning andRACE techniques. The mPRα cDNA sequence consisted of1222base pairsnucleotides, including an open reading frame with1060base pairsnucleotides. This cDNA sequence encoded a precursor protein of353amino acids and the former16amino acids residues formed a signalpeptide. The putative peptide contained13cysteine residues. Its predictedmolecular weight was40.68KDa and the oelectric point was7.08.Comparison of the amino acid sequences and phylogentic analysis showedthat the Oplegnathus fasciatusmPRαhad the close relationship withPerciformes，which was highest similarity with Cynoscion nebulosus (98%).In addition，it had exist7transmembraneregions.The results displayed that the level of mPRα mRNAwas higher in brain,ovary, and lower expression in pituitary, brain, heart, liver, stomach,intestine, spleen, gill, muscle, pyloric caecum, kidney and Head kidney by Real-time Quantitative PCR technique, it was highest in brain. Thereproductive cycle showed that the level of mPRα mRNA in pituitary andovary was maximum in stageⅣof ovarian development and was minimumin stageⅤof ovarian development, however, the level of mPRα mRNA inbrain was maximum in stageⅤof ovarian development.5. Determination of Gonadotropin hormoneinSerum in differentovarian development stage of Oplegnathus fasciatusDetermine the level of Sex steroid hormones in different ovariandevelopment stage of Oplegnathus fasciatus using125I-labeledradioimmunoassay (RIA). The results showed that the content level ofserum FSHrose quickly during yolk formation period an reached its highestlevel, and its level decreased in the period of ovary maturation. The contentlevel of serum LH rose quickly and reached its highest level in the periodof ovary maturation. The result showed that bothof the FSH and LHhadrelationship with ovary development in females.The content of FSH andLH in Serum showed a consistent trend of regular changes with the level ofFSH mRNA and LH mRNA in pituitary. |
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