Cloning And Expression Of Ghrelin Gene In Xiao Meishan Pig's Reproduction Axis

Ghrelin, an acylated 28 amino acid gastric peptide, was isolated from the stomach as an endogenous ligand for growth hormone (GH) secretagogue receptor (GHS-R) in 1999. Circulating Ghrelin is mainly produced by specific cells in the stomach's oxyntic glands.Ghrelin is involved in controlling reproductive functions, and a role for it as a novel regulator of the hypothalamic-pituitary gonadal axis is clearly emerging. In order to research the biological functions of Ghrelin in Xiao Meishan pig's reproductive system,cloning and its expression level during different growth development stages of Xiao Meishan pig's reproduction axis were analyzed.Accroding to Ghrelin gene sequence of Sus scrofa in Genebank, the specific primers for PCR were designed.The Ghrelin gene partial cDNA sequence was amplified by PT-PCR from the pig's stomach, then were cloned into pMD19-T-simple vector and were sequenced. The results showed that the sequence was consisted of 341 nucleotides that crossed the exon.The sequence of amino acid shared 89.3% homology with the published sequence of human,gerbil,mice,rat,dog and 78.6% with cow,sheep and 53.6% with chicken.It established the way for future study on biological function,activity and application of Xiao Meishan pig's Ghrelin gene.According to pigβ-actin cDNA sequence, we designed the primers and regard the PCR productions as the internal standard. Using RT-PCR analysis, Ghrelin mRNA expression could be seen in new born, puberty and sexual maturity male Xiao Meishan pig's hypophysis, hypothalamus, testis, epididymis and new born, puberty and sexual maturity female Xiao Meishan pig's hypophysis, hypothalamus, ovry, uterus. The datas suggested that the gene may play a role in reproduction axis.By fluorescence quantitative PCR (FQ-PCR), the partly mentioned tissues in the text were measured. The results showed in the female pig the hightest expression of Ghrelin mRNA was in sexual maturity pig's ovary and the level in pituitary and hypothalamus were0.449 and 0.316-fold of the ovary.The hightest expression of Ghrelin mRNA in ovary was the sexual maturity's and the level of new born and puberty's were 0.421 and 0.740-fold of the sexual maturity's.