The Porcine Gpr6Gene:Molecule Cloning And Expression Analysis

GPR6is a member of the GPCRs family. Recent studies showed that this gene could play an important role in the maintenance of oocyte meiotic prophase I arrest. The cDNA cloning and functional analysis of GPR6gene not only provides primary information for better understanding of the biological function of GPR6gene in pig, but genetic information for breed improvement as well.In the present study, using homologous sequence alignment approach, combined with reverse transcription polymerase chain reaction (RT-PCR) and3’rapid amplification of cDNA ends (3’RACE), we cloned the cDNA of GPR6. The structure and function of GPR6protein was predicted, and phylogenetic tree of the gene was constructed by bioinformatic methods. Relative expression levels of GPR6mRNA was analysed in various porcine tissues, ovaries of different growth stages, ovaries of different breeds(/hybrids) and follicles of different stages of ovaries by RT-PCR and qRT-PCR. Additionlly, the porcine GPR6protein was expressed in Escherichia coli.1Cloning and sequencing of porcine GPR6geneThe full-length cDNA sequence of GPR6gene of human, mouse and other species were used for homologous sequence alignment. Based on the sequences, the gene specific primers were designed by Primer Premier5for RT-PCR and RACE-PCR, and obtained the sequence of porcine GPR6cDNA (1664bp). The sequence data was submitted to the GenBank databases under accession No. HM777010.2Bioinformatic analysis of GPR6proteinUsing bioinformatics network resources and relevant softwares, we analysed GPR6cDNA which has a1101bp open reading frame (ORF) flanked by a52bp5’UTR and a512bp3’UTR. The putative amino acid sequence has366residues, which contains no signal peptide, but has typical hydrophobic regions and seven transmembrane regions.3Determination of mRNA expression of GPR6gene by Real-time PCRThe relative expression levels of GPR6mRNA in various tissues in hybrid pigs(Duroc x Landrace· Large Yorkshire), ovaries of different growth stages, ovaries of different breeds(/hybrids) and follicles of different stages of ovaries were determined by Real-time PCR. The results showed that GPR6gene was expressed in various tissues, but at very different levels. The expression levels of this gene are higher in the hypothalamus and liver, and lower in oviduct and muscle. The expression levels of this gene are higher in ovaries of hybrid pigs at the age of5and6months than other stages(P<0.01). The mRNA expression of GPR6gene in the ovaries of different pig breeds were no significant differences. The expression of GPR6in large follicles was significantly higher than in the medium follicles and small follicles(P<0.01).4The prokaryotic expression of porcine GPR6The prokaryotic expression vector pET32a-GPR6was constructed by inserting porcine GPR6cDNA into pET32a(+) vector. The sequence of the recombinant was verified by sequencing. The recombinant expression plasmid was transformed into BL21(DE3) and gene expression was induced by administration of IPTG. Protein expression was detected by SDS-PAGE, which shows that the yield of the target protein.