摘要：

为了明确PRKAA1基因在山羊肌内前体脂肪细胞脂质沉积中的作用,通过RT-PCR方法克隆山羊PRKAA1基因的完整CDS区,并开展生物信息学分析;利用双酶切法构建PRKAA1基因过表达载体,并转染山羊肌内前体脂肪细胞。通过实时荧光定量PCR(RT-qPCR)检测PRKAA1基因在幼年期与青年期背最长肌的表达水平,同时检测前体脂肪细胞在诱导分化过程中PRKAA1基因的表达水平及脂代谢相关基因的表达。采用油红O染色法对PRKAA1基因在脂滴形成过程中的作用进行了观察和研究。研究结果表明,山羊PRKAA1基因的核苷酸序列总长为2 520 bp。其中,5'非翻译区(UTR)包含3 bp,编码区(CDS)长达1 680 bp,而3'非翻译区(UTR)则为837 bp,该基因所编码的蛋白质由559个氨基酸残基组成。山羊PRKAA1基因的亲缘关系分析结果显示,绵羊被确定为其最近的亲缘物种,其次是牛、猪、海豚、人、小鼠、大鼠,而鸡是亲缘关系最远的物种。PRKAA1基因在9月龄背最长肌中的表达水平低于2月龄的,诱导分化前体脂肪细胞过程中均存在表达,且在第6天达到最高。在过表达PRKAA1基因的试验中,山羊肌内前体脂肪细胞脂滴含量显著减少。此外,与脂肪代谢相关的基因,如SREBP1-c、FASN、ACC、AGPAT6、PLIN1、ATGL、ACOX1表达均显著下调,且其变化具有统计学意义。然而,DGAT1、DGAT2、LPL和HSL这几个基因的表达水平并未表现出显著变化。这些结果表明,PRKAA1基因的过表达可能通过多种代谢途径影响脂质沉积,具体而言,它能够抑制脂肪酸的从头合成、降低脂肪的合成速率,并提高脂肪的降解速率。这些机制共同作用,从而抑制山羊肌内前体脂肪细胞中的脂质沉积。

关键词: 山羊, PRKAA1, 过表达, 克隆, 脂质沉积

Abstract:

In order to clarify the role of PRKAA1 gene in lipid deposition in goat intramuscular precursor adipocytes,the complete CDS region of the goat PRKAA1 gene was successfully amplified and cloned using RT-PCR,followed by comprehensive bioinformatics analyses. Subsequently,PRKAA1 gene overexpression vectors were constructed utilizing a double enzyme digestion approach and transfected into intramuscular precursor adipocytes.Real-time Fluorescence Quantitative PCR(RT-qPCR)was used to measure the expression level of PRKAA1 gene in the dorsal longissimus dorsi muscle in juvenile and young adults,and the expression level of PRKAA1 gene and lipid metabolism-related genes in precursor adipocytes was also detected in the process of inducing the differentiation of adipocytes. The role of PRKAA1 gene in the formation of lipid droplets was observed and investigated using oil red O staining. The results showed that the total nucleotide sequence length of goat PRKAA1 gene was 2 520 bp. Among them,the 5' untranslated region(UTR)contained 3 bp,the coding region(CDS)was up to 1 680 bp,while the 3' untranslated region(UTR)was 837 bp,and the protein encoded by this gene consisted of 559 amino acid residues. Phylogenetic analysis of the goat PRKAA1 gene revealed that sheep was the most closely related species,followed by cow,pig,dolphin,human,mouse,and rat,whereas chicken was the most distantly related species. The PRKAA1 gene was expressed at a lower level in 9 month-age than 2 month-age in the longest dorsal muscle,and expression was present throughout the induction of differentiation of precursor adipocytes and was at its highest on day 6. In experiments overexpressing the PRKAA1 gene,the content of lipid droplets in precursor adipocytes in goat muscle was significantly reduced. In addition,the expression of genes related to lipid metabolism,such as SREBP1-c,FASN,ACC,AGPAT6,PLIN1,ATGL,and ACOX1 were significantly down-regulated,and the changes were statistically significant. However,the expression levels of several genes,DGAT1,DGAT2,LPL and HSL,did not show significant changes. These results suggest that overexpression of the PRKAA1 gene might affect lipid deposition through multiple metabolic pathways,specifically,it inhibits the ab initio synthesis of fatty acids,decreases the rate of fat synthesis,and increases the rate of fat degradation. These mechanisms work together to inhibit lipid deposition in goat intramuscular precursor adipocytes.

Key words: Goat, PRKAA1, Overexpression, Cloning, Lipid deposition

中图分类号: 

• S827.2