| Abstract Chicken is one of the most important agricultural economic animals.In order to meet the people’s growing demand for chicken products,breeders make high-intensity selection on chicken weight and growth rate,which significantly improves its growth rate and meat yield.However,high-intensity selection brings excessive deposition of abdominal fat,which reduces feed utilization rate and increases feeding cost,Reducing the yield of split meat,reducing the egg production rate,fertilization rate and hatching rate of breeders may induce the occurrence of fatty liver syndrome.The abdominal fat of broilers is discarded as waste during slaughtering and processing,which increases the burden of waste treatment.It is an urgent problem to improve the excessive deposition of abdominal fat in Chinese chickens.Abdominal fat deposition is affected by heredity,nutrition and hormones.However,the research on abdominal fat deposition is not systematic and in-depth.In this study,primary abdominal adipocytes of Gushi chicken were isolated and induced to differentiate into preadipocytes(Ab-pre)and adipocytes(Ab-Ad)for 10 days.The transcriptome of Ab-pre and Ab-Ad groups were sequenced,and the candidate genes related to the proliferation and differentiation of chicken abdominal fat were screened.The function of SLC22A16 was identified.The transcriptional regulation of SLC22A16 on the deposition of chicken abdominal fat was explored at the cellular level by interference and overexpression techniques.1.Screening and identification of key genes for differentiation of abdominal adipocytes in chickenThe primary abdominal adipocytes were isolated from 14 day old Gushi chicken abdominal adipocytes.RNA-seq was performed on abdominal adipocytes before and after differentiation(0d and 10d).A total of 4693 differentially expressed genes were screened,among which 2797 were significantly down regulated,including SCD,INSIG1,ELOVLL5,PPARA,ACSL5 and SOCS3,1896 genes were up-regulated,including SLC22A16,LPIN1,KLF5,COL6A1,FOXO4 and ACOT13.The results of the enrichment analysis of go and KEGG showed that the genes expressed in these differences were enriched in steroid biosynthesis,glycolysis/glycogenesis pathway,ECM receptor interaction,fatty acid biosynthesis and other signal pathways.SLC22A16 was highly expressed in the late stage of abdominal adipocyte differentiation.2.Regulation of SLC22A16 gene on proliferation and differentiation of chicken abdominal adipocytesConstruction of SLC22A16 pcDNA3.1-EGFP overexpression vector was used to transfect chicken primary abdominal preadipocytes for 48 hours.The results of EdU staining and CCK8 test showed that overexpression of SLC22A16 in chicken ICP1 cells significantly promoted cell proliferation and increased cell activity(P<0.05);Oil red O staining showed that the content of lipid droplets increased significantly(P<0.01).The results of EdU staining and CCK8 test showed that interference with SLC22A16 significantly inhibited cell proliferation and decreased cell activity(P<0.05);The results of oil red O staining showed that SLC22A16 was interfered and the content of lipid droplets decreased significantly(P<0.05).After transfection of pcDNA3.1-SLC22A16 into primary preadipocytes for 48 hours,the cells were collected.The results showed that after overexpression of SLC22A16 in primary preadipocytes,the expression of cell proliferation related genes increased(P<0.05).Flow cytometry showed that there were fewer cells in G1 phase and more cells in S phase(P<0.01);Adipocyte differentiation marker genes increased significantly(P<0.05);The content of intracellular triglyceride increased significantly(P<0.01);After transfection of SLC22A16 interference fragment into primary chicken abdominal preadipocytes for 48 hours,the expression of proliferation related genes decreased(P<0.05).Flow cytometry showed that the differentiation marker genes of chicken abdominal preadipocytes inhibited from G1 phase to S phase(P<0.001)decreased significantly(P<0.05);The content of intracellular triglyceride decreased significantly(P<0.05).3.Identification and methylation level of SLC22A16 core promoter in chickenIn this study,different truncated sequences of SLC22A16 gene were cloned in the 5’UTR region.The results of double luciferase reporting system identification indicated that-561 bp～-1074bp of SLC22A16 may have cis-acting elements,such as SP-1,C/EBPα,OCT-1 and other transcription factors.Trans-acting elements may exist in the-1074bp～-1656bp region,such as REV-ErbA,AP-2α and other transcription factors,which provide experimental basis for further studies on the transcriptional regulation of SLC22A16.At the same time,we found CpG islands in the promoter region of-55 bp～-357bp,and then detected the methylation level of SLC22A16 in abdominal preadipocytes on day 0 and day 4 of differentiation by sulfite sequencing.The results showed that the methylation level of SLC22A16 in abdominal preadipocytes on day 0 and day 4 of differentiation was 90%and 20%respectively.In conclusion,SLC22A16 was highly expressed after adipocyte differentiation in Gushi chicken.The results showed that SLC22A16 gene could promote the differentiation of chicken adipocytes by promoting the process of cell cycle,and the differentiation level of SLC22A16 was affected by DNA methylation in promoter region.The above results laid a foundation for clarifying the function of SLC22A16 in chicken abdominal fat deposition. |
