Expression Regulation And Functional Characterization Of FAM20CL Gene In Chicken(Gallus Gallus)

The adipose tissue is the main site of lipid metabolism in mammals,while liver is the main organ of lipid metabolism in poultry.In chicken,more than 90% de novo synthesis of fatty acids is taken place in the liver.Especially in the laying period,liver lipid metabolism is extremely more active than in liver of immature pullets.The development of poultry follicular depends on the induction of estrogen.Under the induction of estrogen,the liver of laying hens synthesizes large amounts of triglycerides,cholesterol and other neutral fats.these fats were then assembled and transported to the growing oocytes to deposit in the yolk.In order to deeply understand the molecular mechanism of lipid metabolism in liver of chicken,our group performed transcriptome data obtained from the liver tissue of Lushi green-shelled hens at 20-week-old pre-laying stage and 30-week-old peak-laying stage.In this study,91 novel genes,which are significantly up-regulated in liver during the peak-laying period,were identified.Among them,FAM20CL（XM₄16256.5）is one of the most significantly up-regulated unannotated genes.To understand the regulatory mechanism and functional characteristics of the FAM20 CL gene,we firstly cloned the coding sequence of FAM20 CL gene and analyzed its origin and evolution by bioinformatics.We then looked into its expression patterns in different tissues and among various developmental stages.Subsequently we explored the regulatory mechanism of the FAM20 CL gene both in vivo and in vitro.Finally,we investigated the biological function of the FAM20 CL gene by using gene over-expression and knockdown technologies.The main results are as followings:1.One unannotated novel gene,FAM20 CL,which was significantly up-regulated in the liver of peak-laying hen,were identified by deep analysis of the transcriptome data obtained from pre-laying chicken and peak laying chicken liver.The coding sequence of FAM20 CL gene was cloned and sequenced using the predicted gene sequence in NCBI as the reference sequence.The CDs of FAM20 CL gene is 1611 bp in length and predicted to encode 256 amino acid protein.Bioinformatics analysis showed that the FAM20 CL gene possess the typical domain of FAM20 C.The phylogenetic tree constructed using the amino acid sequences of FAM20 CL and FAM20 C of different species,indicated that FAM20 CL and FAM20 C originate from the same ancestor,and the FAM20 CL gene only exists in birds and lower vertebrates but not the mammals.2.Using real-time quantitative PCR（q PCR）technology,The spatiotemporal expression patterns of FAM20 CL m RNA in 30-week-old Lushi green-shelled laying hens and in liver at 10 weeks,15 weeks,20 weeks,30 weeks and 35 weeks of Lushi chicken were detected.The results showed that the FAM20 CL m RNA was specifically expressed in liver tissue and differentially expressed in the livers at different developmental stages.The expression level of FAM20 CL in the livers of 30-week-old and 35-week-old（peak-laying period）hens was significantly higher than that in the livers of 10-week-old,15-week-old,and 20-week-old（pre-laying period）hens（P<0.01）.It is speculated that chicken FAM20 CL gene may be related to hepatic lipid metabolism or egg production performance,and the expression of FAM20 CL may be regulated by estrogen.3.The expression changes of FAM20 CL m RANunder different treatment conditions were studied in vivo and in vitro.The results showed that the expression levels of FAM20 CL gene in estrogen-treated young chicken liver and chicken primary hepatocyte were significantly higher than that in control group（P<0.01）.This indicates that the expression of the FAM20 CL gene is regulated by estrogen and it is speculated that it may be involved in estrogen-induced lipid metabolism.4.The FAM20 CL gene overexpression vector pc DNA3.1-FAM20CL-EGFP was successfully constructed,and the FAM20 CL si RNA fragment was designed based on the CDs sequence of the FAM20 CL gene.Overexpression and interference studies were performed.The results showed that the expression level of FAM20 CL gene was significantly up-regulated in the group transfected with the overexpression vector than that in the control group（P<0.01）,and the Apo VLDLII,Apo B,and SCD genes were significantly up-regulated（P<0.05）,while VTG,MTTPL,and PPARγ did not change significantly（P>0.05）.In addition,the concentration of intracellular triglyceride was significantly increased in the treated group（P<0.01）.The expression level of FAM20 CL gene in the group transfected with si RNA fragment was significantly down-regulated（P<0.01）.At the same time,Apo VLDLII,Apo B,VTG and MTTPL genes were significantly down-regulated（P<0.05）,while SCD and PPARγ genes were not significantly changed（P>0.05）.The intracellular triglyceride and total cholesterol levels were significantly reduced in the group transfected with si RNA fragment as well（P<0.01）.This shows that the FAM20 CL gene may be involved in the fat metabolism of chickens.In summary,in this study,the CDs sequence of chicken FAM20 CL gene was cloned and its biological characteristics were analyzed by bioinformatics methods.The FAM20 CL gene was specifically expressed in liver tissue,and its expression was significantly increased with the advent of sexual maturation（P<0.01）.The expression of FAM20 CL gene in chicken liver was regulated by estrogen.The genes related to lipid metabolism were significantly up-regulated（P<0.05）,and the intracellular triglyceride content increased significantly when FAM20 CL gene was overexpressed in the primary hepatocytes（P<0.01）.Meanwhile,the genes related to lipid metabolism were significantly down-regulated（P<0.05）,and the intracellular triglyceride and total cholesterol contents were significantly reduced when the primary hepatocytes were transfected with FAM20 CL gene interference fragments（P<0.01）.This study lays the foundation for defining the function of FAM20 CL gene in chicken lipid metabolism.