山羊FATP2基因的克隆及对前体脂肪细胞脂质沉积的影响

doi:10.11843/j.issn.0366-6964.2023.09.006

摘要/Abstract

摘要： 旨在对山羊脂肪酸转运蛋白2(fatty acid transport protein 2,FATP2)基因进行克隆及生物信息学分析,检测FATP2基因在山羊不同组织中的表达差异,并进一步揭示干扰FATP2基因对山羊肌内前体脂肪细胞脂质代谢的影响。本试验以10月龄健康简州大耳羊(n=12)为试验动物。采用RT-PCR法扩增并克隆山羊FATP2基因,进行序列对比、系统发育树构建及生物信息学分析;利用实时荧光定量PCR检测FATP2基因在山羊不同组织中及在山羊肌内前体脂肪细胞不同分化时期的相对表达水平,合成SI-RNA干扰序列并转染至山羊肌内前体脂肪细胞;使用RT-qPCR检测FATP2干扰效率及脂质代谢相关基因的表达情况;通过Bodipy染色法观察干扰FATP2对脂滴形成的影响,利用GPO-Trinder酶学反应检测甘油三酯含量。结果显示,获得山羊FATP2基因序列全长2 335 bp,CDS区1 863 bp,5'UTR 188 bp, 3'UTR 284 bp; 共编码621个氨基酸,山羊FATP2基因在肝脏表达量最高;RT-qPCR检测结果显示,FATP2表达在细胞中被显著干扰;Bodipy染色结果显示,干扰FATP2基因后脂滴显著减少;甘油三酯测定结果显示,干扰FATP2基因山羊肌内前体脂肪细胞甘油三酯含量显著降低。本研究成功获得山羊FATP2基因CDS区序列,干扰FATP2后山羊肌内前体脂肪细胞脂质沉积显著减少并显著影响脂代谢相关基因的表达,这些结果为进一步阐明FATP2对调控山羊肌内脂肪形成的作用机制提供了重要数据。

关键词: 山羊, FATP2基因, RNA干扰, 肌内前体脂肪细胞

Abstract: The aim of this study was to clone and analyze the fatty acid transport protein 2 (FATP2) gene by bioinformatics in goats, to examine the expression differences of FATP2 gene in different tissues of goats, and to further reveal the effect of interfering FATP2 gene on lipid metabolism in goat intramuscular precursor adipocytes. In this experiment, 10-month-old healthy Jianzhou goats (n=12) were used as experimental animals. The sequences of the FATP2 gene were amplified and cloned by RT-PCR, and sequence alignment, phylogenetic tree construction and bioinformatics analysis were performed. The relative expression levels of FATP2 gene in different tissues of goat and in different differentiation stages of goat intramuscular preadipocytes were detected by real-time fluorescence quantitative PCR. The SI-RNA interference sequence was synthesized and transfected into goat intramuscular preadipocytes. The FATP2 interference efficiency and the expression of the genes related to lipid metabolism were measured by real-time quantitative PCR; The effect of FATP2 interference on lipid droplet formation was observed by Bodipy staining and the triglyceride content was measured by GPO-Trinder enzymatic reaction. The results showed that the full length of the goat FATP2 gene sequence was 2 335 bp, CDS region was 1 863 bp, 5'UTR was 188 bp, 3'UTR was 284 bp, encoding 621 amino acids, and the highest expression of goat FATP2 gene was in the liver. RT-qPCR showed that the expression of FATP2 was interfered significantly in the cells. The Bodipy staining showed that the lipid droplets were significantly reduced after interfering with the FATP2 gene. The results of triglyceride assay showed that the triglyceride content in intramuscular adipocytes of goats was significantly reduced after interfering with FATP2. In this study, the CDS region sequence of goat FATP2 was successfully obtained. After interfering with FATP2, lipid deposition in goat intramuscular preadipocytes was significantly reduced and the expression of lipid metabolism related genes was significantly affected. These results provide important data to further elucidate the mechanism of FATP2's effect on the regulation of intramuscular formation in goats.

Key words: goat, FATP2 gene, RNA interference, intramuscular precursor adipocytes

中图分类号:

S827.2

唐崟梅, 李琪, 李海洋, 林亚秋, 王永, 向华, 黄炼, 朱江江. 山羊FATP2基因的克隆及对前体脂肪细胞脂质沉积的影响[J]. 畜牧兽医学报, 2023, 54(9): 3642-3652.

TANG Yinmei, LI Qi, LI Haiyang, LIN Yaqiu, WANG Yong, XIANG Hua, HUANG Lian, ZHU Jiangjiang. Cloning of the Goat FATP2 Gene and Its Regulatory Effect on Lipid Deposition in Precursor Adipocytes[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(9): 3642-3652.

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