Characterization And Expression Regulation Of NADB-LER Gene Families In Chicken(Gallus Gallus)

Liver is the main organ of chicken fat metabolism,especially for the laying hens.Liver compounds a large amount of lipids andtransported them to the oocytes to develop into the yolk.In order to study the molecular mechanism of lipid metabolism regulation in chickens,We analyzed retrospectively in the liver tissue at the age of 20 weeks old and the age of 30 weeks old,In this study,91 new genes were identified significantly up-regulated in laying hens,after predicting the structure and function of these genes,5 genes,which belonging to the NADB-Rossmann superfamily,were found with the domains of NADP-binding sites,we named them NADB-LER1-5,respectively.On the basis of this,the expression regulation mechanism of chicken NADB-LER1-5 gene families was clarified by bioinformatics analysis tools and molecular biology techniques.Our research laid the foundation for further study on the biological function of NADB-LER1-5 gene family.The main results are as follows:1.Through in-depth analysis of the young chicken and peak laying chicken liver transcriptome data,screening 5 genes in the peak of egg production was significantly increased,and the new gene with a common site of NADP-binding,belonging to the NADB-Rossmann superfamily,named NADB-LER1-5.The CDS sequence of NADB-LER1-5 gene was cloned using the predicted gene sequences in Ensbmel as the reference sequence and PCR primers were designed.The results showed that the full length of NADB-LER1-5 gene was 780 bp,762 bp,780 bp,780 bp and 771 bp,respectively.259,253,259,259,and 256 amino acid residues were encoded,and the phylogenetic tree of NADB-LER1-5 gene family was constructed,which laid the foundation for the study of this new gene family.2.Using real-time fluorescence quantitative PCR(q PCR)technology,NADB-LER1-5 gene expression pattern at the age of 10 weeks old and 30 weeks old in Lushi chicken were detected,and the expression of NADB-LER1-5 in specific tissue at 1 d,1w,10 weeks,15 weeks,20 weeks,30 weeks and 35 weeks of Lushichicken were detected.The results showed that the NADB-LER1-5 gene was widely expressed.NADB-LER1-4 gene were high expressed in lipid metabolism exuberant organs(liver、kidney and duodenum).however,NADB-LER5 gene was high expressed in liver and kidney,also in heart,breast and pancreas.Compared with 10 weeks old chickens,NADB-LER1-4 gene expression of 30 weeks old chickens increased in the liver,a downward trend in the kidney,no changes in the duodenum,and the expression of NADB-LER5 gene increased in the liver,heart and muscle,but in kidney and pancreas showing a downward trend.To investigate the rule of expression of NADB-LER1-5 gene in liver tissue in different developmental stages,the expression level increased significantly in sexual maturity(P <0.05).This showed that the NADB-LER1-5 gene may be associated with hepatic lipid metabolism or directly related with the laying performance,meanwhile,the expression of NADB-LER1-5 gene may be regulated by estrogen in chicken.3.The expression patterns of NADB-LER1-5 under different treatment conditions were analyzed by estrogen treatment with young hens and chicken liver primary cells as models.The results showed that estrogen treatment significantly increased the expression of NADB-LER1-5 in hepatocytes and in vitro cultured primary cells(P <0.05),indicating that NADB-LER1-5 expression was regulated by estrogen,which means NADB-LER1-5 may be involved in estrogen induced lipid metabolism in lipid metabolism.4.In order to further explore the molecular mechanism of how estrogen regulated the expression of chicken NADB-LER1-5 gene,17β estradiol and estrogen receptor antagonist MPP(highly selective antagonist of ER-alpha),ICI 182,780 and tamoxifen(antagonist of ERα and ERβ receptor,and excited GPR30 effect)were used together totreat the primary chick embryo liver cell.The results showed that MPP could antagonize the effect of 17β-estradiol,while ICI182,780 and Tamoxifen have no antagonistic effect on the 17β-estradiol.It is suggested that the effect of estrogen on the expression of NADB-LER1-5 gene was predominantly mediated via ERα in the chicken liver.