Identification And Expression Regulation Of Key Genes In Triglyceride Synthesis Pathway In Chicken (Gallus Gallus)

Triglyceride(TG)synthesis in liver has important influence on production performance,quality of meat and eggs,and other important economic characters in chicken.For a long time,researchers have paid great efforts to investigate the biological process,and made significant progresses in TG synthesis.However,the most of the studies were done in mammals,and the detailed biological process and regulatory mechanism of the TG synthesis pathway in poultry is still not clear until now.In this study,the Lushi Blue-shelled-egg chicken was used as model animals to identify the key genes in triglyceride synthesis pathway and to investigate the regulatory mechanism of the genes in chicken.Firstly,the transcriptome of livers of 20-week juveniles(3 individuals)and 30-week laying hens(3 individuals)were conducted by RNA-seq technology.Then,the significantly differently expressed(SDE)genes between the juveniles and laying hens were screened out.The key genes in chicken TG synthesis pathway were identified by further data mining and q PCR verification.In the meanwhile,the regulatory mechanism of the genes was investigated by a series of in vitro or in vivo experiments.The main results are as follows:1.Glycerol phosphate pathway is the main ways of chicken liver TG synthesis.The genes including glycerol-3-phosphate acyltransferase,mitochondrial(GPAM),acylglycerol-phosphate acyltransferase 2(AGPAT2),acylglycerol-phosphate acyltransferase 3(AGPAT3),lipin 1(LPIN1)and lipin 2(LPIN2)are the key genes in the pathway,and play irreplacable roles in the TG biosynthesis process of liver in chicken.2.The primers for amplifying MGAT and DGAT family genes by PCR were designed according to the sequences predicated in the chicken genome.The coding sequences of chicken LPGAT1 and MOGAT2 genes were cloned and submitted to Gen Bank(Gen Bank ID: KJ628378.1 and KU933271,respectively).Gene expression profile studies showed that the MOGAT1,LPGAT1,SOAT1 and DGAT2 expressed extensively in chicken tissues with relatively higher expression levels in the lipid metabolism organs such as liver,kidney,small intestine(duodenum,jejunum,ileum),except the DGAT2 had a low expression in chicken liver.The MOGAT2 gene only expressed in small intestine(duodenum,jejunum,ileum).3.A new member of the DGAT family,namely DGAT2-like,was identified by the bioinformatic analysis and data mining of the SDE genes in transcriptome data set.The DGAT2-like gene only expressed in chicken liver and kidney,and had a significantly higher expression level in the liver of laying hens than that of pullets.According to the predicted DGAT2-like sequences in the chicken genome,the coding sequence of chicken DGAT2-like gene was cloned(Gen Bank ID: KU853995)for the first time.Comparative sequence analysis revealed that the DGAT2-like had 3 structural domains including LPLAT_MGAT-like domain,DAGAT domain and PLN02783 domain,which could catalyze diacylglycerol(DG)to form TG.Phylogenetic analysis indicated that the chicken DGAT2-like possessed a high consistency with the nucleotide and amino acid sequences of human,mice and rat diacylglycerol acyltransferase 2(DGAT2),originating from a common ancestor.4.To further study the expression regulation mechanism of DGAT2-like gene in liver in chicken,the model of estrogen-treated hens and emboryonic primary hepatocytes were conducted.The expression profiles of DGAT2-like gene with different treatment conditions were detected.The results showed that the expression levels of DGAT2-like in chicken liver,duodenum,kidney and emboryonic primary hepatocytes had no significant changes or exhibited a downtrend.It suggested that the significantly raise of DGAT2-like expression in the egg-laying period was not directly regulated by estrogen.In conclusion,the key genes involved in TG synthesis in liver in chicken were recognized.Meanwhile,a new member of the DGAT family,namely DGAT2-like,was identified.The DGAT2-like gene was highly expressed during the egg-laying period in liver of hens and plays an important role in the TG biosynthesis.This study enriched our understanding of the theory of lipid metabolism in chicken,and paved the way to improve the production performance and quality of meat and eggs for chicken industry.