FATP1对山羊肌内脂肪细胞的促进作用

doi:10.3864/j.issn.0578-1752.2023.10.015

摘要/Abstract

摘要：

【背景】脂肪酸转运蛋白1（FATP1）能够促进哺乳动物脂肪酸摄取，该过程对维持机体脂代谢平衡十分重要，也对家畜的肉质好坏有着重要影响。【目的】通过获得山羊FATP1的CDS区序列，检测该基因在山羊不同组织中的表达量，探究其对山羊肌内脂肪细胞脂质代谢的影响，为进一步揭示FATP1在山羊脂代谢中的作用机制提供参考，为山羊的遗传育种改良提供理论依据。【方法】利用RT-PCR方法克隆获得山羊FATP1的CDS区序列，利用在线工具分析其亲疏水性、跨膜区域、信号肽等生物学特性，并构建其氨基酸序列系统进化树。利用实时荧光定量PCR（RT-qPCR）技术检测FATP1在山羊不同组织中的表达水平，构建其组织表达谱。利用构建的真核表达载体和筛选出的siRNA对山羊肌内脂肪细胞进行FATP1过表达和干扰处理，通过油红O染色和甘油三酯测定检测FATP1过表达和干扰后对山羊肌内脂肪细胞脂质沉积的影响，并通过RT-qPCR技术进一步探究该基因过表达和干扰后对脂质代谢相关基因表达的影响。【结果】克隆获得了FATP1CDS区1 941bp，共编码646个氨基酸残基，预测其分子式为C₃₁₉₆H₅₀₂₆N₈₈₄O₈₉₈S_25，推测该蛋白为碱性疏水稳定蛋白。三级结构预测显示，山羊与绵羊的FATP1蛋白质三级结构相似，而与牛的FATP1蛋白质三级结构略有不同。氨基酸序列系统进化树分析显示，山羊FATP1与绵羊亲缘关系最近。RT-qPCR检测发现FATP1在山羊小肠中表达量最高。油红O染色及甘油三酯测定表明过表达FATP1后山羊肌内脂肪细胞内脂滴数量增多，脂质含量增加，而干扰FATP1后则得到了相反的结果。进一步检测脂质代谢相关基因的表达变化，发现在山羊脂肪细胞中过表达FATP1后，脂肪酸合成、转运等相关基因AGPAT6（P<0.01）、PLIN1（P<0.01）、DGAT2（P<0.01）、FADS2（P<0.01）、FADS1（P<0.01）、ACSL1（P<0.01）及ELOVL3（P<0.05）的表达水平显著升高，而脂解相关基因ACOX1（P<0.01）的表达水平则显著降低；干扰FATP1后，脂肪酸转运、延长等相关基因SCD5（P<0.01）、FABP3（P<0.01）和ELOVL3（P <0.05）的表达量显著下降，脂解相关基因ACOX1（P<0.01）和CPT1B（P<0.05）的表达量显著上升。【结论】FATP1可能通过促进细胞脂质生成相关基因的表达，降低脂质降解相关基因的表达，从而显著促进山羊肌内脂肪细胞脂质的沉积，这些结果为进一步揭示FATP1在调控脂质代谢中的作用及分子机制提供了试验参考。

关键词: 山羊, 脂肪酸转运蛋白1, 肌内脂肪细胞, 脂质沉积

Abstract:

【Background】Fatty acid transporter 1 (FATP1) can promote the uptake of fatty acids in mammals. This process is very important to maintain the balance of lipid metabolism, and also has an important impact on the meat quality of livestock.【Objective】The aim of this study was to obtain the CDS sequence of goat FATP1 gene, to detect the expression of FATP1 gene in different tissues of goats, and to explore its effect on lipid metabolism of goat intramuscular adipocytes, so as to provide a reference for further revealing the mechanism of FATP1 gene in goat lipid metabolism, which can provide a theoretical basis for genetic and breeding improvement of goats.【Method】The CDS of goat FATP1 gene was cloned by real-time fluorescence quantitative PCR (RT-PCR), its biological characteristics, such as hydrophobicity, transmembrane region and signal peptide, were analyzed by online tools, and its amino acid sequence phylogenetic tree was constructed. The expression level of FATP1 gene in different goat tissues was detected by RT-qPCR and its tissue expression pattern was constructed. The constructed eukaryotic expression vector and screened siRNA were used to overexpress and interfere with FATP1 in goat intramuscular adipocytes, the effects of FATP1 gene overexpression and interference on lipid deposition in goat intramuscular adipocytes were detected by oil red O staining and triglyceride determination, and the effects of FATP1 gene overexpression and interference on the expression of genes related to lipid metabolism were further explored by RT-qPCR.【Result】The CDS of FATP1 gene was 1 941 bp, encoding 646 amino acids residues. It was predicted that its molecular formula was C₃₁₉₆H₅₀₂₆N₈₈₄O₈₉₈S₂₅, and the protein was a basic hydrophobic stable protein. Phylogenetic tree analysis of amino acid sequence showed that goat FATP1 was closely related to sheep. RT-qPCR showed that the expression of FATP1 gene was the highest in goat small intestine. Oil red O staining and triglyceride determination showed that the number of lipid droplets and triglyceride content in goat intramuscular adipocytes increased after overexpression of FATP1, but the opposite results were obtained after interference with FATP1. After overexpression of FATP1 in goat adipocytes, the expression levels of fatty acid synthesis, transport and other related genes AGPAT6(P<0.01), PLIN1(P<0.01), DGAT2(P<0.01), FADS2(P<0.01), FADS1(P<0.01), ACSL1(P<0.01) and ELOVL3 (P<0.05) increased significantly, while the expression level of lipolysis related genes ACOX1 (P<0.01) decreased significantly. After interfering FATP1, the expression of fatty acid transport, elongation and other related genes SCD5 (P<0.01), FABP3 (P<0.01) and ELOVL3 (P<0.05) decreased significantly, and the expression of lipolysis related genes ACOX1 (P<0.01) and CPT1B (P<0.05) increased significantly.【Conclusion】FATP1 might significantly promote the lipid deposition of goat intramuscular precursor adipocytes by promoting the expression of genes related to cell lipid production and reducing the expression of genes related to lipolysis, which provided an experimental reference for further revealing the role and molecular mechanism of FATP1 gene in regulating lipid metabolism.

Key words: goat, FATP1, intramuscular adipocytes, lipid deposition

李琪, 杨昌恒, 王永, 林亚秋, 向华, 朱江江. FATP1对山羊肌内脂肪细胞的促进作用[J]. 中国农业科学, 2023, 56(10): 2007-2020.

LI Qi, YANG ChangHeng, WANG Yong, LIN YaQiu, XIANG Hua, ZHU JiangJiang. Role of FATP1 in Promoting Lipid Deposition in Goat Intramuscular Adipocytes[J]. Scientia Agricultura Sinica, 2023, 56(10): 2007-2020.

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https://www.chinaagrisci.com/CN/Y2023/V56/I10/2007

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定量引物"

基因 Gene	全称 Full name	序列 Sequence (5′-3′)	退火温度 Tm (℃)	产物长度 Products length (bp)	基因登录号 Genbank ID
FATP1	Fatty acid transport protein 1	S: ACCACTCGGCAGGGAACATC	60	108	本试验所得序列 The sequence obtained in this experiment
FATP1	Fatty acid transport protein 1	A: TGACGCAATCATCCCAGAAGC	60	108	本试验所得序列 The sequence obtained in this experiment
ACSL1	Acyl-CoA synthetase long-chain family member 1	S: TGACTGTTGCTGGAGACTGG	60	199	XM_005698718
ACSL1	Acyl-CoA synthetase long-chain family member 1	A: CAGCCGTCTTTATCCAGAGC	60	199	XM_005698718
ACSS2	Acyl-CoA synthetase short-chain family member 2	S: GGCGAATGCCTCTACTGCTT	60	100	XM_018057751
ACSS2	Acyl-CoA synthetase short-chain family member 2	A: GGCCAATCTTTTCTCTAATCTGCTT	60	100	XM_018057751
FABP3	Fatty acid binding protein 3	S: GATGAGACCACGGCAGATG	60	120	NM_001285701
FABP3	Fatty acid binding protein 3	A: GTCAACTATTTCCCGCACAAG	60	120	NM_001285701
SCD1	Stearoyl-CoA desaturase 1	S: CCATCGCCTGTGGAGTCAC	60	256	NM_001285619
SCD1	Stearoyl-CoA desaturase 1	A: GTCGGATAAATCTAGCGTAGCA	60	256	NM_001285619
SCD5	Stearoyl-CoA desaturase 5	S: CTGCTCTGGGCCTATTTCTG	60	165	XM_018049650
SCD5	Stearoyl-CoA desaturase 5	A: CCTCGACCACTCGAAGATGT	60	165	XM_018049650
FADS1	Fatty acid desaturase 1	S: GGTGGACTTGGCCTGGATG	60	101	XM_018043055
FADS1	Fatty acid desaturase 1	A: TGACCATGAAGACAAGCCCC	60	101	XM_018043055
FADS2	Fatty acid desaturase 2	S: GCCTGTAGGCTCAGATGTTTGTTC	60	101	XM_018043056
FADS2	Fatty acid desaturase 2	A: TGCCTGGCAGTAACAGAGCAC	60	101	XM_018043056
ELOVL3	ELOVL fatty acid elongase 3	S: CTGTCGGTATCCTGGCTTAT	55	151	XM_005698356
ELOVL3	ELOVL fatty acid elongase 3	A: GCTTTGATCTTGGGAATTATGT	55	151	XM_005698356
ELOVL6	ELOVL fatty acid elongase 6	S: GGAAGCCTTTAGTGCTCTGGTC	60	205	NM_001314257
ELOVL6	ELOVL fatty acid elongase 6	A: ATTGTATCTCCTAGTTCGGGTGC	60	205	NM_001314257
GPAM	Glycerol-3-phosphate acyltransferase, mitochondrial	S: GCAGGTTTATCCAGTATGGCATT	60	64	XM_013975269
GPAM	Glycerol-3-phosphate acyltransferase, mitochondrial	A: GGACTGATATCTTCCTGATCATCTTG	60	64	XM_013975269
AGPAT6	1-acylglycerol-3-phosphate O-acyltransferase 6	A: AAGCAAGTTGCCCATCCTCA	60	101	JI861797.1
AGPAT6	1-acylglycerol-3-phosphate O-acyltransferase 6	S: AAACTGTGGCTCCAATTTCGA	60	101	JI861797.1
LPIN1	Perilipin 1	S: CCCATTGCCAGCACTTCAGA	60	95	XM_018066570
LPIN1	Perilipin 1	A: GCAGCGTACTCGGCAGTATCTC	60	95	XM_018066570
DGAT1	Diacylglycerol O-acyltransferase 1	S: CCACTGGGACCTGAGGTGTC	60	101	XM_018058728
DGAT1	Diacylglycerol O-acyltransferase 1	A: GCATCACCACACACCAATTCA	60	101	XM_018058728
DGAT2	Diacylglycerol O-acyltransferase 2	S: CATGTACACATTCTGCACCGATT	60	100	NM_001314305
DGAT2	Diacylglycerol O-acyltransferase 2	A: TGACCTCCTGCCACCTTTCT	60	100	NM_001314305
ATGL	Adipose triglyceride lipase	S: GGAGCTTATCCAGGCCAATG	60	180	NM_001285739
ATGL	Adipose triglyceride lipase	A: TGCGGGCAGATGTCACTCT	60	180	NM_001285739
HSL	Hormone-sensitive lipase	S: TGCCCAAGACAGAGCCAATG	60	181	EU273879.1
HSL	Hormone-sensitive lipase	A: GCGGAGGAGCCGAGTATCT	60	181	EU273879.1
ACOX1	Acyl-CoA oxidase 1	S: CGAGTTCATTCTCAACAGTCCT	60	245	XM_018063771
ACOX1	Acyl-CoA oxidase 1	A: GCATCTTCAAGTAGCCATTATCC	60	245	XM_018063771
CPT1A	Carnitine palmitoyl transferase 1A	S: TGACGGCTCTGGCACAAGAT	60	164	XM_018043311
CPT1A	Carnitine palmitoyl transferase 1A	A: CGCGAAGTAGTTGCTATTCAC	60	164	XM_018043311
CPT1B	Carnitine palmitoyl transferase 1B	S: ACGAGGAGTCTCACCACTACG	60	111	XM_018048994
CPT1B	Carnitine palmitoyl transferase 1B	A: GTGTGAAGGACTTGTCGAACCA	60	111	XM_018048994
UXT	Ubiquitously expressed transcript	S: GCAAGTGGATTTGGGCTGTAAC	60	180	XM_005700842
UXT	Ubiquitously expressed transcript	A: ATGGAGTCCTTGGTGAGGTTGT	60	180	XM_005700842

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基因 Gene	序列 Sequence (5′-3′)	退火温度 Tm (℃)	产物长度 Products length (bp)	用途 Purpose
FATP1	S: CAAAGCCTGAGGATCTGTGAG	64-55	2078	克隆 Clone
FATP1	A: GGAGTCTTGGGCCAACACA	64-55	2078	克隆 Clone
FATP1	S: CCCAAGCTTGCCACCATGGATTACAAGGAT′ GACGACGATAAGCGGGCTCCAGGTGCAGGT	66-56	1980	亚克隆 Subclone
FATP1	A: CGCGGATCCTCAGAGGGCGAAGGCGCCTGA	66-56	1980	亚克隆 Subclone
S：正义链引物；A：反义链引物。斜体为保护碱基，为酶切位点，为kozak序列，为Flag标签序列 S: Sense primer; A: Antisense primer. Italics are protective bases, enzyme digestion sites, Kozak sequences, Flag tag sequences

基因 Gene	序列 Sequence (5′-3′)
siRNA-NC	UUCUCCGAACGUGUCACGUTT
siRNA-NC	ACGUGACACGUUCGGAGAATT
siRNA-FATP1-1	GGGUCAGUGUCUCAUCUAUTT
siRNA-FATP1-1	AUAGAUGAGACACUGACCCTT
siRNA-FATP1-2	GUGCCACGAACAAGAAGAUTT
siRNA-FATP1-2	AUCUUCUUGUUCGUGGCACTT
siRNA-FATP1-3	CAGGCACCUUCAAGAUUCATT
siRNA-FATP1-3	UGAAUCUUGAAGGUGCCUGTT