<i>FAD3B</i>转基因小鼠肌肉能量代谢的泛素化蛋白组学研究

doi:10.7668/hbnxb.20195968

摘要/Abstract

摘要： 植物体内的FAD3基因是调控多不饱和脂肪酸转化的关键基因,在能量代谢调控方面起重要作用。为了研究FAD3B对小鼠肌肉能量代谢的影响,分别选取FAD3B转基因小鼠(MF)和野生型小鼠(MW)分离腿肌并提取总蛋白,对蛋白样品定性定量检测,采用LC-MS/MS强阳离子柱分离肽段,获得原始质谱数据。利用质谱分析软件处理数据,选取峰值显著的肽段蛋白形成数据集,用转基因小鼠和野生型小鼠的蛋白样品数据做出聚类热图后进行观察分析,再对样品进行生物统计分析和生物信息及功能预测分析,筛选并检测出受FAD3B调控的发生泛素化修饰且与小鼠肌肉能量代谢有关的候选靶蛋白。结果表明:共筛选出180个差异泛素化肽段,且富集于65个蛋白之中。其中,有7个上调肽段富集到了7个蛋白中,有54个下调肽段富集到了31个蛋白中。对所有富集了差异泛素化肽段的靶蛋白进行GO分析、KEGG分析与STRING分析,发现多数靶蛋白与糖酵解、肌肉收缩、蛋白质合成和分解代谢等途径有关。综上,FAD3B基因的外源转入,使得小鼠体内与细胞结构组分、肌肉生长发育、调控能量代谢相关蛋白的泛素化修饰水平发生改变,证明了其除了调控多不饱和脂肪酸的转化外,还可能通过改变关键蛋白的泛素化修饰水平,参与小鼠骨骼肌的生长发育过程,为FAD3B基因调控小鼠骨骼肌生长发育的机制研究提供了新的理论依据与分子靶点。

关键词: 小鼠, FAD3基因, 泛素化蛋白质组学, 泛素化, 生信分析, 肌肉能量代谢

Abstract:

The FAD3 gene in plants plays a crucial role in regulating polyunsaturated fatty acid conversion and energy metabolism.To investigate the impact of FAD3B on muscle energy metabolism in mice,leg muscles were isolated from FAD3B transgenic mice (MF) and wild-type mice (MW),followed by total protein extraction.Protein samples were qualitatively and quantitatively analyzed,and peptides were separated via LC-MS/MS using a strong cation exchange column to obtain raw mass spectrometry data.The data were processed using mass spectrometry software,and a peptide-protein dataset with significant peaks was selected for cluster heatmap visualization to compare MF and MW protein samples.Subsequently,biostatistical and bioinformatics analyses,including functional prediction,were performed to identify candidate target proteins regulated by FAD3B that were ubiquitinated and associated with muscle energy metabolism.The results revealed 180 differentially ubiquitinated peptides enriched in 65 proteins.Among these,seven upregulated peptides were enriched in seven proteins,while 54 downregulated peptides were enriched in 31 proteins.GO analysis,KEGG analysis and STRING analysis were performed on all target proteins enriched with differentially ubiquitinated peptides,and it was found that most of the target proteins were related to glycolysis,muscle contraction,protein synthesis and catabolism.In conclusion,exogenous FAD3B gene transfer alters ubiquitination modification levels in cellular structural components,muscle development,and energy metabolism-related proteins in mice.This demonstrates that FAD3B may influence skeletal muscle growth not only by regulating polyunsaturated fatty acid conversion but also by modulating ubiquitination of key proteins.These results provide a novel theoretical foundation and molecular targets for studying the mechanism of FAD3B in skeletal muscle development.

Key words: Mice, FAD3 gene, Ubiquitinated proteomics, Ubiquitination, Bioinformatics analysis, Muscle energy metabolism

中图分类号:

S813.3生物技术遗传育种

杜新宇, 郭云鹏, 陈卓, 杨欣月, 李新, 居佳雯, 郭益文. FAD3B转基因小鼠肌肉能量代谢的泛素化蛋白组学研究[J]. 华北农学报, 2025, 40(6): 223-230. doi: 10.7668/hbnxb.20195968.

DU Xinyu, GUO Yunpeng, CHEN Zhuo, YANG Xinyue, LI Xin, JU Jiawen, GUO Yiwen. Ubiquitinated Proteomic Study of Muscle Energy Metabolism of FAD3B Transgenic Mice[J]. Acta Agriculturae Boreali-Sinica, 2025, 40(6): 223-230. doi: 10.7668/hbnxb.20195968.

http://www.hbnxb.net/CN/Y2025/V40/I6/223

图/表 5

图1 泛素化蛋白质组学鉴定结果统计学分析 A.考马斯亮蓝染色图;B.转基因小鼠和野生型小鼠泛素化蛋白聚类热图。

Fig.1 Statistical analysis of ubiquitinated proteomics identification results A.Coomassie brilliant blue stain;B.Heat map of MF and MW ubiquitinated proteins clustering.

表1 部分差异肽段的蛋白富集表

Tab.1 Protein enrichment table for some differential peptides

上调差异肽段富集情况 Up-regulated differential peptide enrichment		下调差异肽段富集情况 Down-regulated differential peptide enrichment
蛋白名 Protein name	蛋白功能描述 Protein function description	蛋白名 Protein name	蛋白功能描述 Protein function description
Ttn	肌联蛋白	Mylpf	肌球蛋白调节轻链 11
Neb	星云蛋白	Tpm1	原肌球蛋白 α-1 链
Eef2	真核翻译延伸因子2	Cul5	泛素连接酶
Ube2m	泛素结合酶E2M	Tnni2	肌钙蛋白 I,快速骨骼肌
Acta1	肌动蛋白,α 骨骼肌	Des	结蛋白
Mybpc2	肌球蛋白结合蛋白 C,快型	Gapdh	甘油醛-3-磷酸脱氢酶
Tmem30a	跨膜蛋白30a	Uba1	泛素样修饰剂激活酶 1
		Nedd8	泛素样蛋白
		Itm2c	整合膜蛋白 2C
		Myoz1	肌增生素1

图2 差异表达泛素化蛋白GO富集分析 1.经典糖酵解;2.肌肉收缩;3.肌节组织;4.横纹肌收缩;5.糖酵解过程;6.肌肉收缩的调节;7.心肌收缩;8.糖异生;9.泛素依赖蛋白分解代谢过程;10.蛋白质泛素化;11.横纹肌细丝;12.M带;13.肌节;14.肌原纤维;15.肌球蛋白复合体;16.胞质小核糖体亚基;17.Z盘;18.髓鞘;19.大分子复合体;20.胞质;21.氧转运体活性;22.肌肉的结构成分;23.氧气结合;24.细胞骨架的结构成分;25.泛素结合;26.肌动蛋白纤维结合;27.离子通道结合;28.肌动蛋白结合;29.大分子复合物结合;30.ATP结合。

Fig.2 GO enrichment analysis of differentially expressed ubiquitinated proteins 1.Canonical glycolysis;2.Muscle contraction;3.Sarcomere organization;4.Striated muscle contraction;5.Glycolytic process;6.Regulation of muscle contraction;7.Cardiac muscle contraction;8.Gluconeogenesis;9.Ubiquitin-dependent protein catabolic process;10.Protein ubiquitination;11.Striated muscle thin filament;12.M band;13.Sarcomere;14.Myofibril;15.Myosin complex;16.Cytosolic small ribosomal subunit;17.Z disc;18.Myelin sheath;19.Macromolecular complex;20.Cytosol;21.Oxygen transporter activity;22.Structural constituent of muscle;23.Oxygen binding;24.Structural constituent of cytoskeleton;25.Ubiquitin binding;26.Actin filament binding;27.Ion channel binding;28.Actin binding;29.Macromolecular complex binding;30.ATP binding.

图3 差异表达泛素化蛋白的KEGG富集分析 1.马达蛋白;2.糖异生/糖酵解;3.肥厚型心肌病;4.扩张型心肌病;5.泛素介导的蛋白水解;6.心肌细胞中的肾上腺素能信号传导;7.氨基酸的生物合成;8.疟疾;9.碳代谢;10.非洲锥虫病;11.HIF-1信号通路;12.心律失常性右室心肌病;13.心肌收缩;14.坏死性凋亡;15.帕金森病;16.神经退行性疾病的传播途径-多重疾病;17.卵母细胞减数分裂;18.核苷酸切除修复;19.钙信号通路。

Fig.3 KEGG enrichment analysis of differentially expressed ubiquitinated proteins 1.Motor proteins;2.Glycolysis/Gluconeogenesis;3.Hypertrophic cardiomyopathy;4.Dilated cardiomyopathy;5.Ubiquitin mediated proteolysis;6.Adrenergic signaling in cardiomyocytes;7.Biosynthesis of amino acids;8.Malaria;9.Carbon metabolism;10.African trypanosomiasis;11.HIF-1 signaling pathway;12.Arrhythmogenic right ventricular cardiomyopathy;13.Cardiac muscle contraction;14.Necroptosis;15.Parkinson disease;16.Pathway of neurodegeneration-multiple diseases;17.Oocyte meiosis;18.Nucleotide excision repair;19.Calcium signaling pathway.

图4 差异泛素化蛋白的STRING分析

Fig.4 STRING analysis of differentially ubiquitinated proteins

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FAD3B转基因小鼠肌肉能量代谢的泛素化蛋白组学研究

Ubiquitinated Proteomic Study of Muscle Energy Metabolism of FAD3B Transgenic Mice

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FAD3B转基因小鼠肌肉能量代谢的泛素化蛋白组学研究

Ubiquitinated Proteomic Study of Muscle Energy Metabolism of FAD3B Transgenic Mice

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