The Studies On Transcriptome Analysis Of Adipocytes And The Role Of Relative Functional Genes On Adipogenesis In Yan Yellow Cattle

Beef has become favored increasingly because of its excellent quality of high protein,high unsaturation fatty acid content by consumers.With the improvement of the living quality,consumers are increasingly in demand for beef which is delicious,scculent,and rich in typical high marble pattern.The deposition of intramuscular fat(IMF)is a dynamic balanced process including fat synthesis,fat degradation,and fatty acid transport.The deposition and distribution of intramuscular fat in beef are influenced by many factors such as variety,feeding conditions and feed composition,utilization of molecular biology techniques is an effective way to improve fundamentally carcass quality and fat deposition.Although there are some commercially available cell lines widely used in the market,they can not truly reflect the actual regulatory process of fat synthesis and metabolism in cattle and other large livestock because of their species specificity.In order to further understand the biological characteristics and potential differentiation mechanisms of adipocytes in Yan Yellow cattle,the present study established an in vitro-induced differentiation system,sequenced the transcriptome of different stages of bovine adipocytes by RNA-seq,analysed and validated the candidate genes.The roles of HTR2A and TRIB3 in adipogenic differentiation were further investigated,this research laid the foundation for further study on the mechanism of fat deposition and molecular breeding in yellow cattle.The results were as follows:1.The preadipocytes were isolated by collagenase digestion from newborn calf.After primary and subculturing,they were induced to differentiate into adipocytes by the traditional cocktail method,and the adipocytes were identified.The results showed that:(1)The preadipocyte kept better growth state(in according with the normal growth pattern);(2)The differentiated cells could be stained by oil red O,showing the typical round lipid droplets and the typical characteristics of mature adipocytes;(3)The content of triglyceride changed slowly from day 0 to day 4,significantly increased after day 4;(4)The adipogenic marker PPARy,C/EBPa significantly increased in the early stage of differentiation,and decreased slightly at the end of the differentiation process.2.Transcriptome sequencing of pre-adipogenic differentiation,mid-adipogenic differentiation and late-adipogenic differentiation technology showed that:(1)After the original sequencing data was filtered,high quality data was obtained.The Q20%of clean reads of all these samples reached more than 90%,and the GC content(%)of different stages were in the range of 51%?53%;(2)Compared with Day-0,Day-4 had a total of 2510 differentially expressed genes(DEGs)(1277 up-regulated genes and 1233 down-regulated genes).Compared with Day-4,Day-9 had a total of 2446 DEGs(811 up-regulated genes and 1635 down-regulated genes);(3)GO and KEGG analysis showed that DEGs enriched into more than 2000 functional terms categories and over 300 KEGG pathways,it was found that the early stage of adipocyte differentiation played the most important role in the functional enrichment analysis of different differentiation stages of cattle,and the DEGs mainly involved in cell cycle progression such as chromosome mitosis;The change of cell morphology was mainly at the end of differentiation;(4)The selected randomly DEGs at different stages of differentiation were validated by qRT-PCR.There was slightly different between the quantitative results and RNA-Seq were,but the trends were quite similar,indicating that RNA-seq sequencing results were reliable..3.The positive adipogenic mechanism of candidate gene HTR2A was studied by expression regulation technology in vitro.The results showed that:(1)HTR2A overexpression group could significantly promote the lipid droplets and triglyceride content of mature adipocytes,accelerate the expression of adipogenic key factors C/EBPa and PPARy,while the HTR2A interference was contrary to the HTR2A overexpression in which lipid droplets,triglyceridesthe,and expression of adipogenesis C/EBPa and PPARy expression;(2)HTR2A over-expression could promote significantly the expression of Akt signaling pathway protein and Akt signaling pathway Ser473 phosphorylation protein,accelerate the expression of p38 MAPK signaling pathway protein and p38 MAPK signaling pathway phosphorylation protein,facilitate ERK1/2 MAPK signaling pathway protein and ERK1/2 MAPK signaling pathway phosphorylation protein expression,So it could be suspected that HTR2A might play an important role in promoting adipogenic differentiation through regulating Akt signaling pathway,p38 MAPK signaling pathway and ERK1/2 MAPK signaling pathway.4.The negative adipogenesis mechanism of candidate gene TRIB3 was studied by expression regulation in vitro,the results showed that:(1)The TRIB3 overexpression decreased significantly the lipid droplets and triglyceride content of mature adipocytes,whereas TRIB3 interference,on the contrary,increased lipid droplets and triglyceride content;(2)Overexpression of TRIB3 significantly decreased the expression of Akt signaling pathway protein and Ser473 phosphorylation of Akt signaling pathway,accelerated the expression of phosphorylated protein of p38 MAPK signaling pathway and p38 MAPK signaling pathway,facilitated ERK1/2 MAPK signaling Pathway protein and ERK1/2 MAPK signal pathway phosphorylation protein expression to complete,suggesting that TRIB3 might play a role in the inhibition of adipogenic differentiation of cattle by regulating Akt signaling pathway,p38 MAPK signaling pathway and ERK1/2 MAPK signaling pathway important role.In conclusion,based on the isolation of preadipocytes from newborn Yan Yellow calf,the present study performed transcriptomics analysisof adipocyte at different differentiation stages,and studied the functional genes HTR2A and TRIB3 during adipogenic differentiation.This research would lay the molecular foundation for deepening the understanding of the mechanism of the adipogenic differentiation of the yellow cattle and provide a reference for the mechanism of intramuscular fat deposition.