Study On Mechanism Of DGAT Gene In The Differentiation Of Yanbian Cattle Preadipocytes Induced By Oleic Acid

Eukaryotic cells store neutral lipids in cytoplasmic lipid droplets coated with phospholipids and related proteins.These dynamic organelles are not only the main reservoir of cell energy,but also a part of membrane lipids.Triglyceride (TAG) is the most important form of energy storage in eukaryotic cells.Diacylglycerol acyltransferases (DGAT) catalyze the last step in TAG synthesis by using diacylglycerol and fatty acyl CoA as substrates.Two subtypes of DGAT (DGAT1 and DGAT2) play different roles in mammalian triglyceride metabolism. DGAT1 plays an important role in the regulation of energy metabolism,while DGAT2 is the DGAT enzyme responsible for TAG synthesis and plays a relatively greater role in the synthesis and storage of basic triglycerides.In addition,since the functional activities of several enzymes in the DGAT1 (MOBAT,16 family members) and DGAT2 (MGAT1,7 family members) families are unclear,it is unclear whether tag is produced by enzymes other than DGAT1 and DGAT2.At the same time,it is unclear how DGAT1 and DGAT2 interact and regulate intracellular triglyceride synthesis.In addition,in the previous experiment,we found that the addition of oleic acid can significantly promote the formation of lipid droplets and greatly shorten the differentiation time of preadipocytes.Based on this,the established Yanbian bovine preadipocytes were used as the research model.Oleic acid was added to the differentiation medium as a differentiation promoter.The role of DGAT gene in the regulation of triglyceride and lipid droplet synthesis in the differentiation of preadipocytes for Yanbian cattle was discussed by siRNA interference with DGAT1 and DGAT2 genes and adenovirus mediated overexpression of DGAT2 gene,The main research contents are as follows:1.Bioinformatics and tissue expression analysis of DGAT genes in Yanbian cattleIn this experiment,18 month old ovariectomized Yanbian cattle were used as the research object.The coding sequences of DGAT1 and DGAT2 genes in Yanbian cattle were analyzed by bioinformatics,and the expression levels of 8 different tissue parts were detected at the same time.The results showed that the CDS region sequences of DGAT1 and DGAT2 genes of Yanbian cattle were successfully cloned.The total length of the coding region was 1470 bp and 1086 bp,encoding 489 and 361 amino acids,respectively.The coding region was similar to that of yellow cattle and bison × Yellow cattle,and buffalo have high homology;DGAT1 and DGAT2 are endoplasmic reticulum transmembrane proteins without signal peptides;DGAT1 to α-Spiral and irregular crimp connections are dominant,while DGAT2 is dominant α-Spiral,irregular curl and extended chain connection;The expression levels of DGAT1 and DGAT2 genes were the highest in small intestine and subcutaneous adipose tissue,respectively.The results of this study provide reference for further study on the function of DGAT1 and DGAT2 genes.2.Temporal expression pattern of DGAT gene in preadipocytes of Yanbian cattleIn this experiment,Yanbian cattle preadipocytes were successfully isolated from adipose tissue under the skin of yanbian cattle by collagenase digestion method,and then subcultured and induced differentiation.The results showed that with the prolongation of differentiation time,the expression levels of Pref-1 and DGAT2 genes decreased gradually,while the mRNA expression levels of PPARγ, C/EBP α and DGAT1 genes increased gradually.This experiment provided a good primary cell model for the subsequent studies on the regulation of DGATgene on adipogenic differentiation in Yanbian cattle in vitro.3.Effect of oleic acid on lipid metabolism of Yanbian cattle preadipocytesIn this experiment,different concentrations of oleic acid were added to induce the differentiation of Yanbian cattle preadipocytes.The results showed that with the increase of oleic acid concentration,the formation of lipid droplets,TAG content and adiponectin secretion level could be significantly promoted;When oleic acid was added at 100 μM and 200 μM,there was no significant difference in the expression of related genes in the pathway of triglyceride synthesis of glycerol phosphate (P>0.05).The expression of DGAT1 and DGAT2 genes and proteins in 200 μM test group was significantly higher than that in other test groups (P<0.05).The expression of lipid forming related gene PPAR γ、C/EBP α、FABP4 and PLIN2 increased with the increase of acid concentration,but the expression of SCD gene decreased.It shows that the addition of a certain dose of oleic acid can significantly promote the accumulation of triglycerides in bovine preadipocytes.Based on comprehensive analysis,100 μM was selected as the optimal addition of oleic acid in this test to provide a basic theoretical basis for subsequent tests.4.Study on the mechanism of interfering DGAT gene on TAG and lipid droplet synthesisIn this study,siRNA interference technology targeting DGAT1 and DGAT2 genes was used to detect the effects of inhibition of DGAT1 and DGAT2 genes alone and at the same time on lipid droplet formation and tag synthesis of bovine precursor adipocytes induced by oleic acid.The differentially expressed genes of bovine preadipocytes treated with sh1,sh2,sh12 and shNC(blank vector) were analyzed by transcriptome sequencing (RNA-seq).The results showed that after interfering with DGAT1 and DGAT2 genes,lipid droplets were produced in all experimental groups,but the contents of TAG,adiponectin and lipid droplet area were reduced.The contents of triglyceride in sh-2 and sh-1+2 groups were significantly lower than those in con and sh-NC groups,and the lipid droplet area in sh-2 group was also significantly lower than that in sh-1 group (P<0.05);DGAT1 and DGAT2genes are inhibited either alone or together.The main genes,such as DGAT1,DGAT2,GPAT4 in the phosphoglycerin pathway of triglyceride synthesis and the expression of adipogenesis related genes such as PPAR γ、C/EBP α、SCD was inhibited in varying degrees;RNA-seq results showed that 2070,2242 and 2446 differentially expressed genes were detected in sh1,sh2 and sh12 groups respectively.KEGG analysis of sh2 group showed that the differentially expressed genes in sh2 group were mainly enriched in PPAR signal pathway,AMPK signal pathway and Wnt signal pathway related to adipocyte proliferation and differentiation.At the mRNA level,It shows that DGAT2 gene plays an important role in lipid metabolism.5.Study on the mechanism of overexpression DGAT2 gene on TAG and lipid droplet synthesisIn order to further study the effect of DGAT2gene on TAG and lipid droplet synthesis,Yanbian cattle DGAT2 gene adenovirus overexpression vector was constructed.The differentially expressed genes of bovine preadipocytes transfected with over expressed adenovirus Ad-DGAT2 and Ad-NC (blank vector)were analyzed by transcriptome sequencing.The results showed that overexpression of DGAT2 could significantly increase TAG content and lipid droplet area,and significantly increase lipid related gene PPAR γ、C/EBPα And SCD gene expression (P <0.05),but had no significant effect on the expression of related genes in glycerol phosphate pathway (P> 0.05);A total of 110 differentially expressed genes were detected by RNA-seq,of which 64 were up-regulated and 46 were significantly down regulated;Through go analysis and KEGG analysis,the biological process of differential gene enrichment is mainly related to fatty acid synthesis and lipid metabolism,while the enrichment pathway is mainly in glycerol phospholipid metabolism,polyunsaturated fatty acid synthesis and metabolism and fatty acid biosynthesis.In conclusion,this study preliminarily explored the transcriptional regulation of bovine DGAT gene by constructing siRNA interference fragments of DGAT1 and DGAT2 genes and DGAT2 overexpression adenovirus vector,and then high-throughput sequencing RNA-seq technology was used to study the role of DGAT gene in fat metabolism such as TAG synthesis at the mRNA level,so as to provide an important theoretical basis for the protection,development and utilization of local germplasm resources and the regulation of intramuscular lipid metabolism in beef cattle by means of molecular nutrition.