Study On The Clone Of CDNA Full-Length Sequence,Expression And Tissue Location Of Sika Deer Ghrelin

Ghrelin was discovered in 1999 as a mate of Growth hormone secretagogue receptor.The combination of Ghrelin and GHS-R can promote the secretion of Growth hormone(GH).Researches showed that Ghrelin not only can promote GH secretion but also build up appetite,adjust balance of energy and promote the secretion of gastric acid.Currently,the researches of Ghrelin at home and abroad had mainly focus on human,rodents and livestock,few researches were about sika deer,only the part sequence of sika deer Ghrelin cDNA had obtained by our research group.Therefore,the technologies of RACE,RT-PCR,RT-qPCR and immunochemistry were used to get the cDNA whole sequence of Ghrelin gene of sika deer,and check the expression and location of Ghrelin in animal body.The sequence of 3' RACE and 5' RACE were cloned based on the known fragment of sika deer Ghrelin cDNA.The 539 bp whole sequence of Ghrelin cDNA was obtained firstly through the jointing of the cloned sequence with the known sequence,and the 3'UTR was 128 bp,5'UTR was 46 bp,ORF was 351 bp,the ORF encoded 116 amino acid residue.The results of RT-PCR and RT-qPCR showed that Ghrelin mRNA were expressed in various tissues of sika deer,such as esophagus,rumen,reticulum,omasum,abomasum,duodenum,jejunum,ileum,cecum,colon,liver,pancreas,lung,spleen,kidney,ovary,bladder,heart,ureter,uterus,thymus,lymph gland,endocardium,anterior pituitary,adrenal gland,hypothalamus,tongue and thyroid gland,etc.The expression in abomasum was apparently higher than other organs(P<0.05).Immunohistochemical results showed that Ghrelin-positive cells were widely distributed in the mucosa,submucosa of digestive tract,and also detected in the Muscular layer of esophagus,abomasum,duodenum,cecum,however,few cells were observed in the solid organs(liver,pancreas,lung,spleen,kidney,anterior pituitary,thyroid gland and ovary).The above research resultes had enriched the related biological information of Ghrelin and provided basic information for further studies on Ghrelin physiological functions.