基于转录组和蛋白质组分析筛选银黑狐毛色形成的关键基因

doi:10.11843/j.issn.0366-6964.2025.09.021

摘要/Abstract

摘要：

旨在筛选影响银黑狐毛色形成的关键基因。在相同饲养条件下，本研究选取9月龄、健康且体重相近的雄性银黑狐和北美赤狐各4只，将其分为银黑狐组(YS)和北美赤狐组(CS)。分别采集两组狐的背中部皮肤样本进行转录组测序(RNA-Seq)和串联质量标签(TMT)标记的定量蛋白质组测序分析，筛选差异表达基因(DEGs)和差异表达蛋白(DEPs)并进行功能富集分析，鉴定与银黑狐毛色形成相关的候选基因。结果表明，与北美赤狐相比，在银黑狐皮肤中共鉴定到2 403个差异表达基因(DEGs)和203种差异表达蛋白质(DEPs)。功能富集分析显示，15个DEGs和6个DEPs通过黑色素合成、PI3K-Akt、cAMP、Wnt和MAPK信号通路参与调控黑色素生物合成。转录组与蛋白质组关联分析表明，TYRP1和NRAS等12个基因在mRNA和蛋白质表达水平上呈现一致的显著变化。蛋白互作网络分析进一步确认了TYRP1、NRAS、MLANA、LEF1、TYR、KITLG、EDNRB、GSK3β、AKT2和ERBB3基因为黑色素合成的关键基因。RT-qPCR和Western blot验证了测序结果的可靠性。本研究确定TYRP1和NRAS可能是影响银黑狐毛色表型的关键候选基因，为银黑狐毛色形成的分子调控机制及狐的分子育种提供了理论依据。

关键词: 狐, 毛色, 转录组, 蛋白质组, 关键基因

Abstract:

This study aimed to screen for key genes influencing coat color formation in silver foxes. Under the same feeding conditions, 4 healthy male silver foxes and 4 healthy male red foxes of similar weight at 9 months of age were selected and divided into the silver fox group (YS) and the red fox group (CS). The mid-dorsal skin samples of the two groups were collected for transcriptome sequencing (RNA-Seq) and quantitative proteome sequencing with tandem mass tagging(TMT), and differentially expressed genes (DEGs) and differentially expressed proteins (DEPs) were screened and analysed for functional enrichment, to identify candidate genes related to the formation of coat colour in silver foxes. The results showed that a total of 2 403 differentially expressed genes (DEGs) and 203 differentially expressed proteins (DEPs) were identified in the skin of the silver fox compared with the red fox. Functional enrichment analysis revealed that 15 DEGs and 6 DEPs were involved in melanin biosynthesis and metabolic processes through melanin synthesis, PI3K-Akt, cAMP, Wnt, and MAPK signaling pathways. Correlation analysis between transcriptomic and proteomic data indicated that 12 genes, including TYRP1 and NRAS, exhibited consistent significant changes at both mRNA and protein expression levels. Protein-protein interaction (PPI) network analysis further confirmed that TYRP1, NRAS, MLANA, LEF1, TYR, KITLG, EDNRB, GSK3β, AKT2 and ERBB3 were key genes in melanin synthesis. Validation of the sequencing results was performed using RT-qPCR and Western blot analyses. In this study, TYRP1 and NRAS were identified as possible key candidate genes affecting the coat color phenotype of the silver fox, providing a theoretical basis for the molecular regulatory mechanism of coat color formation in the silver fox and for molecular breeding of foxes.

Key words: fox, coat color, transcriptome, proteome, key genes

中图分类号:

S813.3

李佳鹏, 刘庆, 孙佳钰, 马泽芳, 崔凯. 基于转录组和蛋白质组分析筛选银黑狐毛色形成的关键基因[J]. 畜牧兽医学报, 2025, 56(9): 4379-4392.

LI Jiapeng, LIU Qing, SUN Jiayu, MA Zefang, CUI Kai. Screening of Key Genes for Coat Color Formation in Silver Fox Based on Transcriptome and Proteome Analyses[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(9): 4379-4392.

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基因 Gene	上游引物(5'→3') Forward primer	下游引物(5'→3') Reverse primer	退火温度/℃ Annealing temperature	片段大小/bp Product length
TYRP1	TCTGGCCTCCAGTTACCAACG	AGCAGCAACTACCGCTATGGT	60	131
TYR	GGTACCGGGATCTGCCAATGA	AGGATTCCCGGTTGTGTCCAA	60	146
PMEL	TCTGGCAATGGTCAGCACTCA	TAGCCAGCAACACCAGCAAGA	60	98
MLANA	GGTCACCAGCACAGCAAACTG	TTCTCATAGGGAGGTGGTGCAT	60	80
LEF1	AGCGAATGTCGTAGCTGAGTG	GCTGTCTTTCCTTCCGTGCTA	60	131
GAPDH	GTGACACCCACTCTTCCACCT	GGAGGCCATGTGGACCATGA	60	242

样品 Sample	原始读数/bp Raw reads	质控后读数/bp Clean reads	错误率/% Error rate	%≥Q20	%≥Q30	GC/%	总比对率/% Total mapping
YS-1	46 470 886	44 310 616	0.03	97.48	93.39	48.41	88.94
YS-2	43 981 300	43 098 938	0.03	97.41	93.14	47.96	90.93
YS-3	43 499 072	42 923 648	0.03	97.40	93.00	48.07	92.28
YS-4	44 479 798	43 842 718	0.03	97.55	93.37	47.84	91.81
CS-1	43 101 594	42 518 100	0.03	97.27	92.73	47.71	92.31
CS-2	50 271 118	49 3445 90	0.03	97.47	93.21	47.49	87.01
CS-3	43 156 360	42 4830 92	0.03	97.34	92.92	47.85	93.04
CS-4	43 144 354	42 4230 88	0.03	97.24	92.63	47.45	92.35

GO ID	GO注释GO annotation	P值 P value	差异表达基因DEGs
GO: 0042438	黑色素生物合成过程 melanin biosynthetic process	0.002 04	TYRP1、NRAS、EDNRB、TYR、MFSD12、CTNS、OPN3、OCA2、KITLG、TCF7、EP300、ADCY4、ADCY7、PMEL、LEF1、WNT7B、ADCY5
GO: 0006582	黑色素代谢过程 melanin metabolic process	0.003 08	TYR、MFSD12、CTNS、OPN3、OCA2、APPL1
GO: 0048023	黑色素生物合成过程的正调控 positive regulation of melanin biosynthetic process	0.008 76	TYRP1、OPN3、APPL1
GO: 0048022	黑色素生物合成过程的负调控 negative regulation of melanin biosynthetic process	0.041 59	MFSD12、OPN3
GO: 0042470	黑色素体 melanosome	0.050 00	MLANA、PMEL、ANXA6、MFSD12、CTNS、BACE2、OCA2、APOE

通路ID Pathway ID	通路名称Pathway name	P值P value	差异表达基因DEGs
vvp04916	黑色素合成 Melanogenesis	0.022 23	NRAS、EDNRB、KITLG、ADCY7、ADCY4、WNT7B、EP300、TCF7、LEF1、ADCY5
vvp04024	cAMP信号通路 cAMP signaling pathway	0.007 26	ADCY7、EP300、ADCY5、ASIP、ADCY4、MC1R、EDNRB
vvp04310	Wnt信号通路 Wnt signaling pathway	0.020 08	LEF1、TCF7、WNT7B、EP300
vvp04010	MAPK信号通路 MAPK signaling pathway	0.008 27	NRAS、KITLG、EDNRB、FLT4
vvp04151	PI3K-Akt信号通路 PI3K-Akt signaling pathway	0.006 91	NRAS、KITLG、FLT4、THBS3

通路ID Pathway ID	通路名称Pathway name	差异表达基因DEGs	差异表达蛋白质DEPs
vvp04916	黑色素合成 Melanogenesis	NRAS、EDNRB、KITLG、ADCY5、ADCY7、ADCY4、WNT7B、EP300、TCF7、LEF1	HRNS、NRAS、TYRP1
vvp04024	cAMP信号通路 cAMP signaling pathway	ADCY7、EP300、ADCY5、ASIP、ADCY4、MC1R、EDNRB	AKT2、ERBB3
vvp04310	Wnt信号通路 Wnt signaling pathway	LEF1、TCF7、WNT7B、EP300	GSK3B
vvp04010	MAPK信号通路 MAPK signaling pathway	NRAS、KITLG、EDNRB、FLT4	AKT2、HRAS、NRAS、GSK3B、ERBB3
vvp04151	PI3K-Akt信号通路 PI3K-Akt signaling pathway	NRAS、KITLG、FLT4、THBS3	NRAS、AKT2、HRAS