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

CLC Number: 

• S827.2