Identification And Expression Regulation Of Apela Gene In Chicken (Gallus Gallus)

Apelin is an adipokine involved in the regulation of various biological functions,as a natural ligand,Apelin can regulate fat metabolism by acting on o GPCRs APJ receptor through stimulating downstream pathway in mammals.Recent studies have shown that Apela can also activate the APJ receptor,which plays a similar function with Apelin,and is thought to be a new adipokine.The transcriptome of livers of 20-week juveniles(3 individuals)and 30-week laying hens(3 individuals)were conducted by RNA-seq technology.We found that the expression of Apela is significantly up-regulation at the laying stage.However,the regulation mechanism of the Apela in fat metabolism is not clear yet.In this study,the coding sequence of Apela gene was cloned and analyzed by bioinformatics first.The expression levels of the Apela in different tissues and different development stages were investigated by q PCR then.The regulation mechanism of the Apela was further explored both in vitro and vivo.The main results are as follows:1.The results of q PCR showed that the expression level of chicken Apela gene in laying hens was significantly higher than that in the juveniles(P<0.05),which was consistent with the results of high throughput data.2.The coding sequence of chicken Apela gene was cloned.Amino acid sequence alignment of chicken Apela was couducted by using BLAST tool.The results revealed that the chicken Apela has high similarity to human Apela and rat Apela at the amino acid level.Analysis using SMART tool revealed that Apela has a Low-Complexity Region protein domain,which may be an active unit that binds to the receptor.Evolutionary analysis showed that Apela and Apelin genes originat from a common ancestor and Apela is most likely to be derived from the Apelin.3.Apela was found to be highly expressed in the liver of laying hens according to q PCR.Therefore it is speculated that the expression of Apela gene might be regulated by estrogen and involved in chicken liver lipid synthetically.4.To assess whether the expression of Apela was regulated by estrogen,10 weeks old pullets and embryonic primary hepatocytes were treated with different doses of 17β-estradiol.It turned out that the expression of Apela was significantly up-regulated by 17β-estradiol(P< 0.05).It is speculated that the expression of Apela was regulated by estrogen.5.To further identify which subtype of ERs mediates the expression of Apela gene,embryonic primary hepatocytes were treated with 17β-estradiol combined with different ER subtype antagonists,such as MPP、ICI 182780 and tamoxifen.The expression of Apela was not altered compared with control group,but was significantly decreased(P<0.05)when the hepatocytes were treated with 17β-estradiol combined with tamoxifen or ICI 182780 or MPP compared with 17β-estradiol alone.Because MPP acts as a highly selective antagonist to ERα,whereas tamoxifen and ICI 182780 antagonize nuclear ERs,our results indicate that the estrogen stimulated expression of chicken hepatic Apela is mediated via ER-α.In conclusion,our results suggest that Apela gene highly expressed in the liver,kidney and pancreas of laying hens.Furthermore,the expression of Apela is significantly up-regulation in liver of the laying hens,and its expressions is regulated by estrogen predominantly mediated by ERα.