FTO基因对鸡肌内脂肪细胞脂沉积的调控作用

doi:10.3864/j.issn.0578-1752.2025.22.017

摘要/Abstract

摘要：

【目的】脂肪量和肥胖相关基因（FTO）作为与人类和畜禽脂肪沉积密切相关的基因，其在鸡肌内脂肪沉积中的作用尚未见相关报道。通过明确 FTO 对鸡肌内脂肪细胞增殖及脂滴沉积的影响，筛选响应其调控的关键通路与基因，为解析鸡肌内脂肪沉积分子调控网络提供理论依据。【方法】构建FTO慢病毒表达载体，外源转染FTO慢病毒表达载体和阴性对照载体至鸡原代肌内脂肪细胞，利用荧光倒置显微镜观察荧光，实时荧光定量PCR（qPCR）检测转染组和阴性对照组FTO mRNA的表达变化，确保FTO慢病毒表达载体成功转染至细胞；FTO慢病毒表达载体转染72 h和96 h后，CCK8试剂盒检测转染组和阴性对照组细胞增殖变化；转染后6 d,油红O染色，异丙醇萃取脂滴，检测肌内脂肪细胞脂滴沉积的变化，甘油检测试剂盒检测培养基中甘油含量变化；通过转录组测序、生物信息学分析及蛋白互作（PPI）分析，筛选响应FTO调控肌内脂肪细胞脂沉积的关键通路和基因。【结果】FTO慢病毒表达载体转染至肌内脂肪细胞，qPCR结果表明，与对照组相比，转染组FTO mRNA表达极显著升高（P<0.01）,表明FTO慢病毒表达载体成功转染至细胞；转染FTO慢病毒表达载体72和96 h 后，与对照组相比，肌内脂肪细胞的增殖能力显著降低（P<0.05）；FTO慢病毒表达载体转染肌内脂肪细胞6 d后，转染组细胞脂滴沉积能力显著高于阴性对照组（P<0.05），培养基中的甘油含量显著低于阴性对照组（P<0.05）。转录组测序结果表明：共筛选到164个差异表达基因响应FTO调控肌内脂肪沉积，其中上调基因71个，下调基因93个；差异表达基因KEGG富集分析结果显示，共有13信号通路显著富集，其中黏着斑，肌动蛋白细胞骨架的调控，丝裂原活化蛋白激酶信号通路，间隙连接，细胞因子 - 细胞因子受体相互作用, 叉头盒蛋白 O 信号通路, 细胞凋亡, C 型凝集素受体信号通路, 转化生长因子 -β 信号通路(P<0.05)通路与脂肪代谢相关；进一步筛选响应FTO调控的关键通路和基因，将显著富集通路里的差异基因做PPI分析，结果显示 PIK3R2, FGF16, FGF9和RHOA为响应FTO调控鸡肌内脂肪细胞脂沉积的关键基因，显著富集通路——肌动蛋白细胞骨架调控通路为核心通路。【结论】FTO具有抑制鸡肌内脂肪细胞增殖、促进脂沉积、抑制脂肪分解的作用；PIK3R2、FGF16、FGF9和 RHOA（肌动蛋白细胞骨架的调控通路）是响应FTO调控肌内脂肪细胞脂沉积的关键基因/通路。

关键词: 鸡, FTO, 肌内脂肪

Abstract:

【Objective】 The fat mass and obesity-associated gene (FTO) is closely related to fat deposition in humans and livestock/poultry. However, its role in intramuscular fat deposition in chickens has not been reported yet. This study aims to clarify the effect of FTO on the proliferation and lipid droplet deposition of chicken intramuscular adipocytes, screen the key pathways and genes responsive to its regulation, and provide a theoretical basis for analyzing the molecular regulatory network of intramuscular fat deposition in chickens. 【Method】In this study, an FTO lentiviral expression vector was constructed, and chicken primary intramuscular adipocytes were transfected with the FTO lentiviral expression vector and a negative control vector. Fluorescence was observed using a fluorescence inverted microscope, and quantitative PCR (QPCR) was performed to detect changes in FTO gene expression in the transfection and negative control groups, thus ensuring successful transfection of the FTO lentiviral expression vector into the cells. Cell proliferation changes in the negative control and transfection groups were then detected using a CCK8 assay at 72 h and 96 h post-transfection with the FTO lentiviral expression vector. At the sixth day of transfection, oil red O staining was performed, lipid droplets were extracted with isopropanol to detect changes in intramuscular adipocyte lipid droplet deposition, and a glycerol assay kit was used to detect changes in glycerol content in the culture medium. Finally, key pathways and genes responding to FTO regulation of intramuscular adipocyte lipid deposition were screened through transcriptome sequencing, bioinformatics analysis, and PPI analysis. 【Result】Following transfection of intramuscular adipocytes with the FTO lentiviral expression vector, QPCR analysis revealed a significant increase in FTO mRNA expression in the transfected group compared with the control group (P<0.01), confirming successful transfection of the FTO lentiviral expression vector into the cells. The proliferation capacity of intramuscular adipocytes was significantly reduced at 72 h and 96 h post-transfection with the FTO lentiviral expression vector, compared with the control group (P<0.05). After six days of transfecting intramuscular adipocytes with the FTO lentiviral expression vector, the transfected group exhibited a significantly higher capacity for lipid droplet deposition than the negative control group (P<0.05), while the glycerol content in the culture medium was significantly lower (P<0.05). Transcriptome sequencing identified a total of 164 differentially expressed genes responding to FTO-regulated intramuscular fat deposition, with 71 genes upregulated and 93 downregulated. KEGG enrichment analysis of these differentially expressed genes revealed significant enrichment in 13 signaling pathways, including focal adhesion, regulation of actin cytoskeleton, MAPK signaling pathway, gap junction, cytokine-cytokine receptor interaction, FoxO signaling pathway, apoptosis, C-type lectin receptor signaling pathway, and TGF-beta signaling pathway (P<0.05), all of which were directly or indirectly related to lipid metabolism. Key pathways and genes responsive to FTO regulation were further screened, and PPI (Protein-Protein Interaction) analysis on the differentially expressed genes in significantly enriched pathways was performed. The results showed that PIK3R2, FGF16, FGF9, RHOA and NGF were key genes for FTO-regulated intramuscular adipocyte lipid deposition in chickens. Among them, PIK3R2, FGF16, FGF9, and RHOA were all enriched in the regulation of actin cytoskeleton pathway, which was identified as the core pathway. 【Conclusion】 The FTO gene inhibited intramuscular adipocyte proliferation, promoted lipid deposition, and suppressed lipolysis in chickens. PIK3R2, FGF16, FGF9 and RHOA (regulation of actin cytoskeleton pathway) have been identified as key genes/pathways that responded to FTO regulation of intramuscular adipocyte lipid deposition.

Key words: chicken, FTO, intramuscular fat

黄华云, 隋雨乐, 孔熠, 梁忠, 杨苗苗, 刘星, 韩威. FTO基因对鸡肌内脂肪细胞脂沉积的调控作用[J]. 中国农业科学, 2025, 58(22): 4786-4796.

HUANG HuaYun, SUI YuLe, KONG Yi, LIANG Zhong, YANG MiaoMiao, LIU Xing, HAN Wei. Regulatory Effect of FTO Gene on Lipid Deposition in Chicken Intramuscular Adipocytes[J]. Scientia Agricultura Sinica, 2025, 58(22): 4786-4796.

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基因 Genes	引物序列 Primer sequence	长度 Length (bp)
β-actin	F:5′-gtccaccttccagcagatgt-3′	169
β-actin	R:5′-ataaagccatgccaatctcg-3′	169
FTO	F:5′-ctggtcctccaagaagttcg-3′	159
FTO	R:5′-ctgctcttctggcaagctct-3′	159

通路 Pathway	P值P value	基因 Genes
MAPK 信号通路 MAPK signaling pathway	0.014	HGF，FGF9，PDGFB，NGF，AKT3，FGF16
细胞因子 - 细胞因子受体相互作用Cytokine-cytokine receptor interaction	0.018
叉头盒蛋白 O 信号通路 FoxO signaling pathway	0.014	CDKN2B，CDKN2A，PIK3R2，AKT3，FOXO4
转化生长因子 -β 信号通路 TGF-beta signaling pathway	0.031	CDKN2B，CDKN2A，SMAD6，RHOA
黏着斑信号通路 Focal adhesion pathway	0.003	HGF，ITGA1，RHOA，PDGFB，PIK3R2，AKT3
肌动蛋白细胞骨架的调控 Regulation of actin cytoskeleton	0.003	ITGA1，FGF9，RHOA，PDGFB，PIK3R2，FGF16
间隙连接Gap junction	0.001	TUBA4A，HTR2B，TUBB4B，PDGFB，GJA1
细胞凋亡Apoptosis	0.016	TUBA4A，NGF，PIK3R2，AKT3
Toll 样受体信号通路 Toll-like receptor signaling pathway	0.026	TLR5，PIK3R2，AKT3
C 型凝集素受体信号通路 C-type lectin receptor signaling pathway	0.029	RHOA，PIK3R2，AKT3
沙门氏感染Salmonella infection	0.002	TLR5，IFNGR2，IL18，RHOG
甲型流感Influenza A	0.019	IFNGR2，IL18，PIK3R2，AKT3
糖尿病并发症中的 AGE-RAGE 信号通路 AGE-RAGE signaling pathway in diabetic complications	0.031	PIM1，PIK3R2，AKT3