山羊GPR35基因表达特性分析及对皮下脂肪细胞分化作用的研究

doi:10.11843/j.issn.0366-6964.2023.12.011

Abstract

Abstract: The purpose of this study was to clone the GPR35 gene sequence of goat, explore its expression characteristics and elucidate its effect on the differentiation of goat subcutaneous preadipocytes and its possible mechanism. In this study, the complete sequence coding for amino acids in protein (CDS) of GPR35 gene in one-year-old healthy male Jianzhou Big ear goats (n=4) were cloned by RT-PCR and analyzed by bioinformatics. Quantitative real-time PCR (qRT-PCR) was used to detect the expression level of GPR35 gene in goat various tissues and goat subcutaneous adipocytes at different differentiation stages. Then GPR35 overexpression vector (pEGFP-N1-GPR35) and its interference sequence (Si-GPR35) were transfected into goat subcutaneous adipocytes and induced by oleic acid. The changes of lipid droplets were observed by Oil Red O staining and Bodipy staining, and the changes of triglyceride content were detected. Finally, qRT-PCR was used to detect the changes in the expression of marker genes related to adipocyte differentiation, triglyceride synthesis and decomposition (all samples have 3 biological sample replicates and 3 technical replicates). The results showed that the cloned goat GPR35 gene sequence was 1 167 bp and the open reading frame was 906 bp, encoding 301 amino acid residues. The expression of GPR35 gene in longissimus dorsi was significantly higher than that in other muscle tissues of goats (P<0.01); Its expression in subcutaneous fat content was significantly higher than that in other adipose tissues (P<0.01); It was significantly higher in spleen than that in other internal organs of goats (P<0.01). In addition to that, the expression of GPR35 gene was the highest in subcutaneous adipose cells at 120 h after induction (P<0.01). Overexpression and interference of GPR35 inhibited and promoted the content and aggregation of lipid droplets in subcutaneous adipocytes, respectively, and the relative content of triglyceride was significantly decreased and increased, respectively. The expression of C/EBPα, C/EBPβ, PPARγ, AP2 and SREBP1 genes was significantly down-regulated (P<0.01), and the expression of HSL was significantly up-regulated (P<0.01) after overexpressing GPR35 gene. However, the expression of C/EBPα, C/EBPβ, AP2, SREBP1 and DGAT1 genes was significantly up-regulated (P<0.01), and the expression of FASN was significantly down-regulated (P<0.01) after silencing GPR35 gene. These results suggest that GPR35 gene may be a negative regulator during the differentiation of goat subcutaneous preadipocytes, which may be achieved by regulating the expression of C/EBPα, C/EBPβ, AP2, SREBP1 or interacting with CXCL17 protein.

Key words: goat, GPR35, bioinformatics analysis, subcutaneous adipocytes, differentiation of cells

CLC Number:

S827.2

MENG Qiuchi, LU Guangyu, CHEN Dingshuang, LIN Yaqiu, WANG Ruilong, ZHONG Chaosong, WANG Yong, LIU Wei, WANG Youli, LI Yanyan, LI Zhixiong. Goats GPR35 Gene Expression Characteristics Analysis and the Effect on Subcutaneous Fat Cell Differentiation[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(12): 4993-5007.

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Goats GPR35 Gene Expression Characteristics Analysis and the Effect on Subcutaneous Fat Cell Differentiation

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