Molecular Cloning, Expression And Sequence Analysis Of GnRH In Sebastes Schlegelii And Cynoglossus Semilaevis

Gonadotropin-releasing hormone (GnRH) is a decapeptide neurohormone that plays avital role in hypothalamus-pituitary-gonad axis (HPG). Black rockfish (Sebastesschlegelii) is an important cultured fish in North China which has the advantages ofstrong disease resistance, larger individual, growing fast and ovoviviparous.Half-smooth tongue sole (Cynoglossus semilaevis) is a large precious marineeconomic fish in the offshore waters of our country, and has many strong points suchas fast-growing in aquaculture. To study types, distribution and roles of GnRHs andits receptor in the two species can provide theoretical basis for elucidating theregulation mechanism of GnRH on HPG axis.In this study, based on the homologous cloning and RACE techniques, the full-lengthcDNA of three types GnRH in S. schlegelii and one type GnRH in C. semilaevis wereobtained. Using bioinformatic softwares and quantitative real-time PCR to study eachof them, respectively:A. The full-length S. schlegelii GnRH-I cDNA was424bp, composed of61bp of5’UTR,288bp of ORF, and36bp of3’UTR, and it could encode96aa residues withan estimated molecular mass of10.4kDa. This propeptide included a signal peptideof22aa residues. qPCR analysis indicated that GnRH-I was expressed in all the ninetissues of adult S. schlegelii, with a highest level in the whole brain and ovary;B. The full-length S. schlegelii GnRH-II cDNA was615bp, composed of146bp of5’UTR,258bp of ORF, and184bp of3’UTR, and it could encode86aa residues with an estimated molecular mass of9.6kDa. This propeptide included a signalpeptide of23aa residues. qPCR analysis indicated that GnRH-II was only expressedin the whole brain, gonads, heart, spleen, muscle and intestine of adult S. schlegelii,with a highest level in the whole brain and ovary;C. The full-length S. schlegelii GnRH-III cDNA was506bp, composed of45bp of5’UTR,273bp of ORF, and156bp of3’UTR, and it could encode91aa residues with an estimated molecular mass of10.1kDa. This propeptide included a signalpeptide of23aa residues. qPCR analysis indicated that GnRH-III was only expressedin the whole brain and gonads of adult S. schlegelii;In addition, qPCR analysis indicated all the three S. schlegelii GnRH genes expressionhad a larger fluctuation during development.D. The full-length C. semilaevis GnRH-III cDNA was344bp, composed of36bp of5’UTR,276bp of ORF, and32bp of3’UTR, and it could encode92aa residues withan estimated molecular mass of10.3kDa. This propeptide included a signal peptideof23aa residues. qPCR analysis indicated that GnRH-III was expressed only in brainand gonads in adult C. semilaevis; the GnRH-III transcript was maternally depositedwith a larger fluctuation pre and post embryo hatching.Bioinformatic softwares predication indicated all the three types of S. schlegeliiGnRH and one type of C. semilaevis GnRH were secreted protein. The GnRHprecursor amino acids sequence alignment among the two fishes and other teleostswere remarkedly similar, indicating the highly conservation of teleost GnRH inevolution. The tissue expression patterns revealed all these GnRHs obtained abovewere highly expressed in the relative organs of reproduction axis, suggesting theymay play important functions in the reproduction, and may participate in the wholereproductive process as well.(2) A2359bp GnRHR-I cDNA was obtained from S. schlegelii, composed of304bpof5’UTR,1281bp of ORF, and774bp of3’UTR, and it could encode426aaresidues. Funtion domain predication suggested that GnRHR-I of S. schlegeliipossesses a domain of7transmembrane helixes and belonged to G protein-coupledreceptor (GPCR) family, having the classical characteristics of GPCR: anextracellular N-terminal tail,3ECLs,3ICLs,7transmembraneα-helixes connected byECLs and ICLs, and an intracellular C-terminal tail. qPCR analysis showed that inadult male and female S. schlegelii, GnRHR-I mRNA had high level expression in thewhole brain and gonads, but could not be detected in kidney and spleen and had lowlevel expression in other six tissues.(3) The promoters of the three types GnRH of S. schlegelii were cloned by genome walking and were analyzed by bioinformatic softwares: The promoters of GnRH1,GnRH2, and GnRH3were4k bp,3.5k bp and2.5k bp in length, respectively. Acharacterize TATA box was found at31bp upstream from the transcription start site(TSS) of GnRH1and GnRH3, and a characterize GC box were found at80bpupstream from the TSS of GnRH2. There were putative binding sites for Sp1, NF-1,Pit-1, C/EBP, CREB, Oct-1, GATA-1, Brn, USF and MyoD from the promoter regionsof all the three GnRH genes. Binding sites of the nuclear receptors, including GR, AR,ER, PR, RAR and RXR were also predicted, suggesting that these motifs play similarkey roles in GnRH regulation. However, the presence of binding sites for TR andOlf-1could only be confirmed in GnRH1and GnRH2, but not in GnRH3, suggestingthe regulations of these transcription factors on GnRH were type-specific. Theseresults showed that the three types of GnRH genes in S. schlegelii were differentiallyregulated and had distinct physiological roles.(4) The S. schlegelii GnRH-III peptide (without signal peptide) was expressed in theform of fusion protein using pET32a as expression vectors and BL21(DE3) pLysS asexpression host. SDS-PAGE analysis showed an expression protein band of27kDacorresponding to the recombinant mature peptide. The further analysis proved that agreat part of the recombinant mature peptide existed as soluble type.