| In the process of high-end beef production,long-term fattening leads to excessive accumulation of cheap fat in subcutaneous and abdominal parts of beef cattle,which reduces feed returns and feeding economic benefits.Increasing the economic value of cheap fat is an effective means to solve this problem.Bovine adipose tissue contains a large amount of saturated fatty acids,which can easily lead to cardiovascular and cerebrovascular diseases.Unsaturated fatty acids can reduce the incidence of cardiovascular and cerebrovascular diseases,prevent inflammation and cancer,and participate in immune regulation.Therefore,the economic value of cheap fat can be improved by increasing the content of unsaturated fatty acids in adipose tissue,and breeding new beef cattle varieties with this excellent property can fundamentally solve the problem of resource waste in the production of high-grade beef.However,the mechanism of metabolite formation in bovine adipose tissue is still unclear,and the candidate genes controlling unsaturated fatty acids are not clear enough.To solve this problem,the changes of metabolites in bovine preadipocytes were analyzed by ultra-high performance liquid chromatoc-tandem mass spectrometry(UPLC-MS/MS).At the same time,the gene expression map was constructed from the transcriptome level.The metabolic and transcriptome data were integrated to establish the regulatory network of gene expression on metabolites,and the main candidate gene FADS2 was selected to regulate the content of unsaturated fatty acids.Finally,a cell model of stable overexpression of FADS2 was constructed using lentivirus as a carrier,and the mechanism of FADS2 regulation on the production of unsaturated fatty acids in bovine adipocytes was revealed.The specific research results are as follows:(1)Changes in different metabolites during lipogenic differentiation of bovine precursor adipocytesOil red O staining was performed on bovine preadipocytes at 0,2,4 and 8 days of induction,and it was found under microscope that the lipid droplets increased from zero to more with the passage of time,with the largest lipid droplets at the 8th day of induction.A total of 962 metabolites were detected in the cells collected at these four stages through extensive targeted metabolome sequencing analysis,which were divided into 22 types of substances,among which amino acids and their metabolites accounted for 27 %,and fatty acylates,glycerides and glycerophospholipids accounted for 14 %.Hierarchical cluster analysis results showed that amino acids and fatty acid metabolites continuously accumulated over time.A total of 397 differential metabolites were screened from six comparison groups in four stages of pairwise comparison.The results of time-series expression analysis of differential metabolites showed that the metabolites consistent with the trend of lipid droplet phenotypic change were mainly amino acids,fatty acyl,glycerol and phospholipids.They are mainly significantly enriched in unsaturated fatty acid biosynthesis,amino acid biosynthesis and arachidonic acid metabolic pathway.(2)Construction of gene expression profiles during lipogenic differentiation of bovine precursor adipocytesBovine precursor adipose cells at different stages of lipid differentiation(0,2,4 and 8days)were collected,RNA was extracted,and transcriptomic sequencing was performed.A total of 20,125 genes were detected.After further screening,a total of 6,714 differentially expressed genes were obtained from the six comparison groups.In the first 4 days of lipogenic differentiation,the biological processes are mainly cell division,the cell cycle of mitosis and the biosynthesis of cholesterol,and in the 4 days after lipogenic differentiation,the biological processes are mainly the transcription of RNA polymerase II promoter.In addition,KEGG results showed that the differentially expressed genes of the six comparison groups were significantly co-enriched in PPAR signaling pathway,MAPK signaling pathway,unsaturated fatty acid biosynthesis,amino acid biosynthesis,and insulin signaling pathway.In addition,RT-q PCR was used to verify the expression of 8 differential genes,which was consistent with the transcriptome sequencing results.(3)Construction of a gene regulatory network for metabolites during lipogenic differentiation of bovine precursor adipocytesIn this study,through the correlation analysis of genes and metabolites,it was found that the correlation between differential genes and differential metabolites became more and more significant over time,and the main correlation was positive.The expression trend was further analyzed by combining the change multiples of genes and metabolites.Among the genes and metabolites with absolute correlation values greater than 0.8,the number of genes and metabolites with up-regulated expression was significantly higher than that of genes and metabolites with down-regulated expression.Next,through KEGG pathway coenrichment analysis,it was found that 6714 differential genes and 397 differential metabolites were significantly co-enriched in unsaturated fatty acid biosynthesis,amino acid biosynthesis,alanine,aspartate and glutamate metabolic pathways.(4)Candidate gene mining for the regulation of unsaturated fatty acid content during lipogenic differentiation of bovine precursor adipocytesCanonical correlation analysis(CCA)was used to explore candidate genes regulating unsaturated fatty acid content,and FADS2,ACOT7 and ACOT2(ENSBTAG00000046814)were found to be candidate genes affecting unsaturated fatty acid content.Among them,FADS2 has the highest correlation with arachidonic acid,eicosapentaenoic acid,linoleic acid and oleic acid,which has been paid more attention in subsequent studies.(5)Validation of the effect of candidate gene FADS2 on unsaturated fatty acids and its regulatory mechanismThe lentivirus overexpressing FADS2 was successfully packaged by constructing a three-plasmid lentivirus packaging system.The above lentivirus was used to infect C3H10T1/2 cell line,and the positive rate of C3H10 T1/2 cell line of FADS2 reached 96.5 %,and the gene expression level of FADS2 was more than 2607 times higher than that of the control group(P < 0.01).The FADS2 mesenchymal stem cell model was successfully constructed.The results of oil red O staining showed that,Compared with the control group,the adiposedeposition capacity of FADS2 MSCS was significantly improved.RT-q PCR was used to study the effect of overexpression of FADS2 on the expression of key genes in fat deposition.The results showed that: Key fat deposition genes ACC1,FABP4,FASN,GPDH,LPL,PPARG,SCD1 and unsaturated fatty acid biosynthesis pathway gene HACD2 were significantly up-regulated in FADS2 overexpressing mesenchymal stem cells.In addition,oleic acid,linoleic acid and palmitoleic acid were detected by gas chromatograph,and it was found that the contents of oleic acid and palmitoleic acid in FADS2 group were increased by 18 % and 25.3 %,respectively,compared with the control group.Linoleic acid could be detected in FADS2 group,while it was not detected in the control group because its content did not reach the detection threshold.In summary,based on metabolomics and transcriptomic analysis,this study found that FADS2 may be a candidate gene affecting the content of unsaturated fatty acids,and successfully constructed a mesenchymal stem cell model with overexpression of FADS2 to further explore the influence of overexpression of FADS2 on the expression of key genes of fat deposition and the content of unsaturated fatty acids.The mechanism of FADS2 regulating the production of unsaturated fatty acids in bovine precursor adipocytes was revealed,which provided theoretical and technical support for improving the lipogenesis ability of bovine precursor adipocytes and improving the economic value of cheap adipocytes,and provided necessary information and resources for scientific research of bovine precursor adipocytes and breeding new beef cattle varieties with such excellent characteristics. |
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