过表达DGAT2基因对延边牛前体脂肪细胞分化的影响

doi:10.16431/j.cnki.1671-7236.2023.08.002

摘要/Abstract

摘要： 【目的】探究二酰基甘油酰基转移酶2(DGAT2)对牛前体脂肪细胞分化的影响及其对脂质代谢相关信号通路的调控作用。【方法】利用ADV1穿梭质粒构建包含目的基因的重组穿梭质粒,将重组穿梭质粒与骨架质粒pGP-Ad-Pac载体共转染293A细胞,制得过表达腺病毒载体Ad-DGAT2,用Ad-DGAT2与Ad-NC(阴性对照组)感染牛前体脂肪细胞;用油酸诱导分化96 h后,利用油红O染色观察脂滴生成情况,并检测甘油三酯(TAG)和脂联素(ADP)含量;通过实时荧光定量PCR检测脂肪生成相关基因表达情况;最后对其进行转录组测序,筛选差异表达基因并进行GO功能注释和KEGG通路富集分析。【结果】过表达DGAT2基因可显著增加脂滴数量及TAG、ADP含量(P＜0.05),可显著上调磷酸甘油途径DGAT1、甘油磷酸酰基转移酶4(GPAT4)、酰甘油磷酸酯酰基转移酶4(AGPAT4)以及脂质代谢途径中过氧化物酶体增殖活化受体γ(PPARγ)、脂肪分化相关蛋白(PLIN2)、CCAAT/增强子结合蛋白α(C/EBPα)和硬脂酰辅酶 A 去饱和酶(SCD)的mRNA表达水平(P＜0.05)。转录组测序结果显示,与Ad-NC组相比,Ad-DGAT2感染的前体脂肪细胞中共筛选出110个差异表达基因,其中64个基因上调,46个基因下调。KEGG通路富集分析显示,差异表达基因主要富集在PPAR信号通路、甘油磷脂代谢、脂肪酸生物合成和AMPK信号通路中。【结论】过表达DGAT2基因可以促进延边牛脂肪细胞中脂滴形成、TAG积累和相关成脂基因的表达,转录组测序筛选出了与脂肪代谢相关的差异表达基因。本研究结果表明DGAT2基因在前体脂肪细胞分化过程中发挥了重要调控作用,为延边牛的分子育种提供了重要的理论依据。

关键词: DGAT2基因; 甘油三酯; 前体脂肪细胞; 过表达; 转录组

Abstract: 【Objective】 The purpose of this study was to investigate the effects of diacylglycerol acyltransferase 2 (DGAT2) on differentiation of bovine preadipocytes and its regulation of lipid metabolity-related signaling pathways.【Method】 The recombinant shuttle plasmid containing the target gene was constructed using the ADV1 shuttle plasmid,and then 293A cells were co-transfected with the recombinant shuttle plasmid and the skeleton plasmid pGP-Ad-Pac vector,and Adenovirus overexpressed vector Ad-DGAT2 was prepared.Bovine preadipocytes were infected with Ad-DGAT2 and Ad-NC (negative control group).96 h after differentiation induced by oleic acid,lipid droplet formation was observed by Oil red O staining,and the contents of triglyceride (TAG) and adiponectin (ADP) were detected.The expression of genes related to lipogenesis was detected by Real-time quantitative PCR.Finally,transcriptome sequencing was performed to screen differentially expressed genes,and GO functional annotation and KEGG pathway enrichment analysis were performed.【Result】 Overexpression of DGAT2 gene significantly increased the number of lipid droplets and the contents of TAG and ADP (P<0.05).Meanwhile,overexpression of DGAT2 could significantly up-regulate mRNA expression of DGAT1,glycerol phosphate acyltransferase 4 (GPAT4) and acylglycerol phosphate acyltransferase 4 (AGPAT4) in phosphoglycerol pathway and peroxisome roliferatoractivated receptor gamma (PPARγ),perilipin 2(PLIN2),CCAAT/enhancer binding protein α (C/EBPα) and stearoyl-COA desaturase (SCD) in lipid metabolism pathway (P＜0.05).Transcriptome sequencing results showed that a total of 110 differentially expressed genes were screened from preadipocytes infected with Ad-DGAT2 compared with Ad-NC group,among which 64 genes were up-regulated and 46 genes were down-regulated.KEGG pathway enrichment analysis showed that differentially expressed genes were mainly concentrated in the PPAR signaling pathway,glycerol phospholipid metabolism,fatty acid biosynthesis and AMPK signaling pathway.【Conclusion】 Overexpression of DGAT2 gene could promote lipid droplet formation,TAG accumulation and the expression of related adipogenic genes in Yanbian bovine adipocytes.Transcriptional sequencing screened out differentially expressed genes related to fat metabolism.The results indicated that DGAT2 gene played an important regulatory role in the differentiation of precursor adipocytes,which provided an important theoretical basis for molecular breeding of Yanbian bovine.

Key words: DGAT2 gene; triacylglycerol; preadipocytes; overexpression; transcriptome

中图分类号:

郭盼盼, 李强, 李香子, 严昌国, 金鑫. 过表达DGAT2基因对延边牛前体脂肪细胞分化的影响[J]. 中国畜牧兽医, 2023, 50(8): 3045-3055.

GUO Panpan, LI Qiang, LI Xiangzi, YAN Changguo, JIN Xin. Effect of Overexpression of DGAT2 Gene on Differentiation of Yanbian Bovine Preadipocytes[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(8): 3045-3055.

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