灵丘青背山羊ACSL1基因克隆、生物信息学分析及其在脂肪细胞分化过程中的表达研究

doi:10.16431/j.cnki.1671-7236.2025.02.001

摘要/Abstract

摘要： 【目的】克隆灵丘青背山羊长链脂酰辅酶A合成酶1(long-chain acyl-CoA synthetase 1，ACSL1)基因CDS区序列并进行生物信息学分析，探究ACSL1基因在灵丘青背山羊不同组织及皮下脂肪细胞分化过程中的表达规律。【方法】根据GenBank中山羊ACSL1基因序列(登录号：XM_018041882.1)设计特异性引物，以灵丘青背山羊肝脏组织cDNA为模板，通过PCR扩增ACSL1基因CDS区序列并克隆、测序，与其他物种进行相似性比对及系统进化树构建，并采用在线软件对ACSL1蛋白进行生物信息学分析。利用实时荧光定量PCR方法检测ACSL1基因在灵丘青背山羊不同组织以及ACSL1、ACACA、PPARγ、FASN基因在皮下脂肪细胞不同分化时期的表达情况。【结果】灵丘青背山羊ACSL1基因CDS区序列全长2 100 bp，编码699个氨基酸。相似性比对发现，灵丘青背山羊ACSL1蛋白氨基酸序列与绵羊的相似性最高，达99.0%。系统进化树显示，灵丘青背山羊与绵羊的亲缘关系最近，与虎鲸的亲缘关系最远，且在不同物种进化过程中具有高度保守性。生物信息学分析发现，灵丘青背山羊ACSL1蛋白分子式为C₃₅₂₉H₅₅₆₇N₉₂₉O₁₀₀₀S₃₆，分子质量为78.2 ku，理论等电点为7.46，半衰期为30 h，不稳定系数为32.61。ACSL1蛋白为碱性疏水性蛋白，存在1个跨膜结构，无信号肽。ACSL1蛋白有49个磷酸化位点，主要在线粒体中发挥生物学功能。灵丘青背山羊ACSL1蛋白二级结构由α-螺旋(39.63%)、无规则卷曲(31.33%)、延伸链(20.74%)及β-转角(8.30%)组成。ACSL1蛋白与FASN、ACACA、LPL等10个蛋白可能存在互作。组织表达分析发现，ACSL1基因在灵丘青背山羊各组织中均有表达，其中在肝脏中表达量最高，且显著高于其他组织(P＜0.05)，在皮下脂肪、肾脏和心脏中表达量次之。检测山羊脂肪细胞不同分化时期基因的时序表达，结果显示，与分化第0天相比，山羊ACSL1基因表达量在分化第8天达到峰值(P＜0.05)；ACACA和FASN基因表达量均在分化第10天达到峰值(P＜0.05)；PPARγ基因表达量在分化第6天达到峰值(P＜0.05)。【结论】本研究成功克隆了灵丘青背山羊ACSL1基因CDS区序列，并明确了其在山羊不同组织和脂肪细胞分化过程中的表达规律。ACSL1基因在肝脏和皮下脂肪组织中有较高的表达，ACSL1、ACACA、PPARγ、FASN基因表达量在脂肪细胞诱导分化过程中均呈现先升后降的趋势，研究结果为进一步探究ACSL1基因在山羊脂肪沉积过程中的分子机制提供了理论依据。

关键词: 灵丘青背山羊; ACSL1基因; 克隆; 生物信息学; 组织表达; 脂肪细胞分化

Abstract: 【Objective】 This study was aimed to clone the CDS sequence of long-chain acyl-CoA synthetase 1 (ACSL1) gene in Lingqiu Qingbei goats and carry out bioinformatics analysis,and explore the expression pattern of ACSL1 gene in different tissues and the differentiation of subcutaneous fat cells in Lingqiu Qingbei goats. 【Method】 Based on ACSL1 gene sequence of Capra hircus in GenBank (accession No.:XM_018041882.1),specific primers were designed.The CDS sequence of ACSL1 gene was amplified by PCR using the cDNA template of liver in Lingqiu Qingbei goats and cloned and sequenced.The similarity alignment and phylogenetic tree construction were carried out with other species,and the bioinformatics analysis of ACSL1 protein was performed by the online software.The expression of ACSL1 gene in different tissues in Lingqiu Qingbei goats and ACSL1,ACACA,PPARγ and FASN genes at different differentiation stages of subcutaneous fat cells in goats were analyzed by Real-time quantitative PCR. 【Result】 The CDS region of ACSL1 gene in Lingqiu Qingbei goats was 2 100 bp in length,which encoded 699 amino acids.Similarity alignment revealed that the amino acid sequence of ACSL1 protein in Lingqiu Qingbei goats had the highest similarity with Ovis aries,reaching 99.0%.The results of phylogenetic tree showed that Lingqiu Qingbei goats had the closest relationship with Ovis aries and the farthest relationship with Orcinus orca,and the evolutionary process was highly conservative in different species.Bioinformatics analysis showed that the molecular formula of ACSL1 protein in Lingqiu Qingbei goats was C₃₅₂₉H₅₅₆₇N₉₂₉O₁₀₀₀S₃₆,the theoretical molecular weight was 78.2 ku,the theoretical isoelectric point was 7.46,the half-life was 30 h,and the instability coefficient was 31.61.ACSL1 protein was an alkaline hydrophobic protein with a transmembrane structure and no signal peptide.ACSL1 protein had 49 phosphorylation sites,and mainly played a biological function in mitochondria.The secondary structure of ACSL1 protein in Lingqiu Qingbei goats was composed of alpha helix (39.63%),random coil (31.33%),extended chain (20.74%) and beta turn (8.30%).ACSL1 protein might interact with 10 proteins such as FASN,ACACA and LPL.The tissue expression analysis showed that ACSL1 gene was expressed in all tissues of Lingqiu Qingbei goats,with the highest expression in liver,which was significantly higher than that in other tissues (P＜0.05),followed by subcutaneous fat,kidney and heart.The results of temporal expression of genes in goat adipocytes at different differentiation stages showed that,compared with day 0 of differentiation,the expression of ACSL1 gene in goats reached the peak on the 8th day of differentiation (P＜0.05),the expression of ACACA and FASN genes reached the peak on the 10th day of differentiation (P＜0.05),and the expression of PPARγ gene reached the peak on the 6th day of differentiation (P＜0.05). 【Conclusion】 The CDS sequence of ACSL1 gene in Lingqiu Qingbei goats was successfully cloned，and the expression pattern of ACSL1 gene in different tissues and the differentiation of subcutaneous fat cells of goats were clarified.The expression of ACSL1 gene was high in liver and subcutaneous fat.In the process of adipocyte differentiation,the expression of ACSL1,ACACA,PPARγ and FASN genes showed a trend of increasing first and then decreasing.The results provided a theoretical basis for further exploring the molecular mechanism of ACSL1 gene in fat deposition of goats.

Key words: Lingqiu Qingbei goats; ACSL1 gene; cloning; bioinformatics; tissue expression; adipocyte differentiation

中图分类号:

S827
Q78

艾小楠, 程俐芬, 白璞, 陈正灏, 薛丽娜, 周胜花. 灵丘青背山羊ACSL1基因克隆、生物信息学分析及其在脂肪细胞分化过程中的表达研究[J]. 中国畜牧兽医, 2025, 52(2): 499-511.

AI Xiaonan, CHENG Lifen, BAI Pu, CHEN Zhenghao, XUE Lina, ZHOU Shenghua. Cloning and Bioinformatics Analysis of ACSL1 Gene and Its Expression Pattern During Adipocyte Differentiation in Lingqiu Qingbei Goats[J]. China Animal Husbandry and Veterinary Medicine, 2025, 52(2): 499-511.

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