基于转录组测序分析湖羊波浪形羔皮形成的关键基因

doi:10.11686/cyxb2025290

摘要/Abstract

摘要：

旨在对湖羊4/4直毛和4/4波浪形羔皮进行转录组测序，以分析影响羔皮性状的关键基因。采集4/4直毛和4/4波浪形羔皮羊的体侧部皮肤组织（n=4），提取总RNA进行转录组测序，筛选差异表达基因（DEGs），进行基因本体论（GO）和京都基因与基因组百科全书（KEGG）富集分析，并进一步开展KEGG通路的基因集富集分析（GSEA）及DEGs的转录因子预测。对7个DEGs进行实时荧光定量PCR验证（qRT-PCR），同时比较DEGs在不同花案面积个体羔皮直毛和波浪处的表达。结果显示，4/4直毛和4/4波浪形羔皮中共筛选出315个DEGs（阈值|log₂FC|≥1且FDR<0.05），其中波浪形羔皮中274个基因下调，41个基因上调。GO分析发现，DEGs主要富集在细胞外结构组织、细胞外基质和整合素结合等3771个条目；KEGG分析显示，DEGs主要富集在细胞黏附分子、PI3K-AKT信号通路和RAS信号通路等209条通路。GSEA显示，DEGs主要与组氨酸代谢、肾素分泌和蛋白质消化吸收等通路相关；与4/4直毛形羔皮相比，富集到的与毛囊有关的RAS、PI3K-AKT和RAP1信号通路在4/4波浪形羔皮中呈下调趋势；转录因子预测发现编码转录因子（TFs）的差异基因共12个，其中上调基因2个，下调基因10个，主要包括SRY相关高迁移率族盒（SOX）、叉头框（FOX）和T盒（T-box）等TFs家族。qRT-PCR结果显示，筛选出的7个与羔皮性状相关的基因表达趋势与转录组测序结果一致，且IGF1和MFAP5基因在不同花案面积个体羔皮波浪处的表达量显著低于直毛处。综上，筛选出的AEBP1、IGF1、MMP27、MFAP5、RPTN、SPI1和THY1可作为影响羔皮性状的重要候选基因，为进一步解析湖羊波浪形羔皮形成的分子机制奠定基础。

关键词: 湖羊, 羔皮, 毛囊发育, 羊毛弯曲, 转录组测序

Abstract:

The aim of this study was to conduct transcriptome sequencing on Hu lambskins with 4/4 straight hairs and 4/4 wavy pattern， and to identify the key genes affecting lambskin traits. Skin tissues （n=4） were collected from the side of the body of 4/4 straight hair and 4/4 wavy pattern Hu lambs. Total RNA was extracted for transcriptome sequencing， and differentially expressed genes （DEGs） were screened and subjected to gene ontology （GO） and kyoto encyclopedia of genes and genomes （KEGG） enrichment analyses. Gene set enrichment analysis （GSEA） of the KEGG pathways was also conducted， and the DEGs encoding transcription factors （TFs） were identified. Seven DEGs were selected for verification of their transcript profiles by real-time fluorescence quantitative PCR （qRT-PCR）， and the transcript profiles of DEGs in the site of straight hair and wavy pattern in individual lambskin with different wavy areas were compared. Results showed that a total of 315 DEGs （threshold |log₂FC|≥1 and FDR<0.05） were screened out by comparing the transcriptomes of 4/4 straight hair and 4/4 wavy pattern lambskins. Of the 315 DEGs， 274 were down-regulated and 41 were up-regulated in wavy pattern lambskin compared with straight hair lambskin. The GO analysis revealed that DEGs were mainly enriched in 3771 items including extracellular structural tissues， extracellular matrix， and integrin binding. The KEGG analysis showed that DEGs were mainly enriched in 209 pathways including the cell adhesion molecules， PI3K-AKT signaling， and RAS signaling pathways. The GSEA results indicated that DEGs were mainly related to pathways such as histidine metabolism， renin secretion， and protein digestion and absorption. The enriched signaling pathways related to hair follicles， including the RAS， PI3K-AKT， and RAP1 signaling pathways， showed a down-regulated trend in the 4/4 wavy pattern lambskin compared with the 4/4 straight hair lambskin. Among the DEGs， 12 encoded TFs， of which two were up-regulated and 10 were down-regulated in wavy pattern lambskin compared with straight hair lambskin. The down-regulated TF DEGs included those encoding SRY-related high-mobility group box （SOX）， forkhead box （FOX）， and T-box （T-box） TFs. The transcript levels of the seven candidate genes determined by qRT-PCR were consistent with those determined from the transcriptome analysis， and confirmed significantly lower transcript levels of IGF1 and MFAP5 in wavy pattern skin regions than in straight hair skin regions in individuals with different pattern coverage areas. In conclusion， AEBP1， IGF1， MMP27， MFAP5， RPTN， SPI1， and THY1 were identified as important candidate genes affecting lambskin traits. These results lay a foundation for further understanding the molecular mechanism of the formation of wavy lambskin in Hu sheep.

Key words: Hu sheep, lambskin, hair follicle development, wool crimp, transcriptome sequencing

谢婧, 孟旭铭, 仓振, 华云涛, 周贤毅, 茆达干. 基于转录组测序分析湖羊波浪形羔皮形成的关键基因[J]. 草业学报, 2026, 35(7): 188-200.

Jing XIE, Xu-ming MENG, Zhen CANG, Yun-tao HUA, Xian-yi ZHOU, Da-gan MAO. Analysis of key genes in Hu wavy pattern lambskin based on transcriptome sequencing[J]. Acta Prataculturae Sinica, 2026, 35(7): 188-200.

图/表 9

表1 实时荧光定量PCR引物序列

Table 1 qRT-PCR primer sequences

基因 Gene	名称 Name	基因库编号 Genebank number	引物序列 Primer sequence （5′-3′）	产物长度 Product length （bp）
AEBP1	AE结合蛋白1AE binding protein 1	XM_012176971.5	F： CTCATGCAATACCTGTGCCG	142
AEBP1	AE结合蛋白1AE binding protein 1	XM_012176971.5	R： TCCCAAACTCTGAGCCCATC	142
IGF1	胰岛素样生长因子1Insulin-like growth factor 1	XM_027965761.2	F： TTCTATCTGGCCCTGTGCTTG	138
IGF1	胰岛素样生长因子1Insulin-like growth factor 1	XM_027965761.2	R： CCCCGTGGGCTTGTTGAAATA	138
MMP27	基质金属肽酶27Matrix metallopeptidase 27	XM_004015972.5	F： TGCTGTCTTGCCAGATTATCCC	149
MMP27	基质金属肽酶27Matrix metallopeptidase 27	XM_004015972.5	R： GGGTCACTTCATCATACCTCCAG	149
MFAP5	微纤维相关蛋白5Microfibril-associated protein 5	XM_015094961.3	F： TCAAGCCTTCTACCGATGACC	70
MFAP5	微纤维相关蛋白5Microfibril-associated protein 5	XM_015094961.3	R： TTCTCATCCCGGCACTCTGT	70
RPTN	重复素Repetin	XM_060396929.1	F： AGAATGGGGACTCCACCTCA	93
RPTN	重复素Repetin	XM_060396929.1	R： CTGGGTCATTTGGTCTCCTGA	93
SPI1	Spi-1原癌基因Spi-1 proto-oncogene	XM_027979753.3	F： CAACGCCAAACGCACGAATA	71
SPI1	Spi-1原癌基因Spi-1 proto-oncogene	XM_027979753.3	R： TCCCAGTAATGGTCGCTGTG	71
THY1	Thy-1细胞表面抗原Thy-1 cell surface antigen	XM_060399793.1	F： CCTCCTGCTGACAGTCTTACA	132
THY1	Thy-1细胞表面抗原Thy-1 cell surface antigen	XM_060399793.1	R： AACTCATACTGCATGGGCGT	132
GAPDH	甘油醛-3-磷酸脱氢酶Glyceraldehyde-3-phosphate dehydrogenase	NM_001190390.1	F： TCGGAGTGAACGGATTTGGC	76
GAPDH	甘油醛-3-磷酸脱氢酶Glyceraldehyde-3-phosphate dehydrogenase	NM_001190390.1	R： ACGATGTCCACTTTGCCAGT	76

图 1 直毛和波浪形羔皮组织形态学特性P：初级毛囊Primary hair follicle； S：次级毛囊Secondary hair follicle.

Fig.1 Histomorphological characteristics of straight hair and wavy pattern lambskins

表2 直毛和波浪形羔皮毛囊比较

Table 2 Comparison of hair follicles between straight hair and wavy pattern lambskins

组别

Group

次级毛囊数量/初级毛囊数量Number of secondary hair follicles/number of primary hair follicles （S/P）

初级毛囊直径Primary hair follicle diameter （PD， μm）

次级毛囊直径Secondary hair follicle diameter （SD， μm）

4/4直毛 4/4 straight hair

4.24±0.25a

176.65±6.84a

60.05±2.84a

4/4波浪4/4 wavy pattern

2.67±0.25b

138.21±6.50b

45.55±3.56b

表3 数据质控表

Table 3 Data quality control sheet

样本名称Sample	原始测序读数Raw reads （bp）	过滤后读数Clean reads （bp）	Q20 （%）	Q30 （%）	GC含量GC content （%）
S₁	41094942	41094936	99.40	97.18	48.65
S₂	40508630	40508628	99.40	97.25	48.60
S₃	46929166	46924146	99.53	97.87	48.88
S₄	40044654	40044652	99.54	97.95	48.63
W₁	41862838	41859348	99.51	97.71	48.47
W₂	43925888	43925882	99.48	97.60	48.88
W₃	40125986	40125986	99.49	97.67	47.44
W₄	42378798	42375874	99.45	97.48	47.76

图 2 湖羊4/4直毛和4/4波浪形羔皮的差异表达基因A：差异表达基因的火山图Volcano map of differentially expressed genes； B：差异表达基因聚类分析图Cluster analysis diagram of differentially expressed genes. S：直毛Straight hair； W：波浪Wavy pattern. Padj：校正后P值Adjusted P-value； FC：倍数变化Fold change. 下同The same below.

Fig.2 Differentially expressed genes in Hu lambskins with 4/4 straight hair and 4/4 wavy pattern

图3 差异表达基因的GO和KEGG通路富集分析A： GO富集分析GO enrichment analysis； B： KEGG富集分析KEGG enrichment analysis.

Fig.3 GO and KEGG pathway enrichment analysis of differentially expressed genes

图4 磷脂酰肌醇-3-激酶-蛋白激酶B信号通路相关差异基因聚类热图A：生成磷脂酰肌醇3-激酶IA型的PI3K-AKT信号通路途径The PI3K-AKT signaling pathway for generating phosphatidylinositol 3-kinase type IA； B：生成磷脂酰肌醇3-激酶IB型的PI3K-AKT信号通路途径The PI3K-AKT signaling pathway for generating phosphatidylinositol 3-kinase type IB.

Fig.4 Phosphatidylinositol-3-kinase-protein kinase B signaling pathway-related differential gene clustering heatmap

图5 KEGG通路基因集富集分析结果

Fig.5 Gene set enrichment analysis （GSEA） results of KEGG pathway

图6 实时荧光定量PCR检测候选基因表达量A： 4/4直毛和4/4波浪形羔皮7个候选基因表达Expression of seven candidate genes lambskins with 4/4 straight hairs and 4/4 wavy pattern； B：不同波浪面积羔皮个体直毛与波浪处IGF1和MFAP5的表达Expression of IGF1 and MFAP5 at straight hair site and wavy pattern site in individual lambskins with different wavy areas. *： P<0.05； **： P<0.01.

Fig.6 Expression of candidate genes detected by real-time quantitative PCR

参考文献 41

[1]	Li Z N， Zhang Z L， Du J Z， et al. Morphological investigation on the formation of skin wavy pattern in the Huyang lambs. Scientia Agricultura Sinica， 1982， 15（3）： 73-82.
	李志农，张仲倫，杜建中，等. 湖羊羔皮花纹形成的形态学观察. 中国农业科学， 1982， 15（3）： 73-82.
[2]	Chen R H. Identification and grading of Hu lambskin. Journal of Economic Animal， 1980（1）： 40-41.
	程瑞禾. 小湖羊皮的鉴定与分级. 毛皮动物饲养， 1980（1）： 40-41.
[3]	Chen F X.Tanning technology of chekiang lamb skin. China Leather， 2005， 34（5）： 40， 46.
	程凤侠. 小湖羊皮鞣制工艺. 中国皮革， 2005， 34（5）： 40， 46.
[4]	Prost-Squarcioni C. Histology of skin and hair follicle. Médecine Sciences， 2006， 22（2）： 131-137.
[5]	Sun W， Ni R， Yin J F， et al. Genome array of hair follicle genes in lambskin with different patterns. PLoS One， 2013， 8（7）： e68840.
[6]	He M L， Lv X Y， Cao X K， et al. SOX18 promotes the proliferation of dermal papilla cells via the Wnt/beta-catenin signaling pathway. International Journal of Molecular Sciences， 2023， 24（23）： 16672.
[7]	Li X B， Liu Z F， Liu Y， et al. Mining genes related to wool bending of Zhongwei Goat based on WGCNA and GSEA. Acta Veterinaria et Zootechnica Sinica， 2022， 53（9）： 2930-2943.
	李晓波，刘占发，刘悦，等. 基于WGCNA与GSEA方法挖掘调控中卫山羊羊毛弯曲相关基因. 畜牧兽医学报， 2022， 53（9）： 2930-2943.
[8]	Yu L J， Zhang Y H， Lazate·Ainiwaer， et al. Screening microRNAs related to wool crimp in Chinese Merino sheep. Xinjiang Agricultural Sciences， 2021， 58（3）： 573-580.
	于丽娟，张艳花，拉扎特·艾尼瓦尔，等. 中国美利奴羊羊毛弯曲相关microRNA的筛选. 新疆农业科学， 2021， 58（3）： 573-580.
[9]	Dong Y J， Hao X J， Wu J Q， et al. Exploration of the effect of SHH on wool bending through Krox20 regulation of IGFBP5 expression based on sheep keratinocytes. Acta Veterinaria et Zootechnica Sinica， 2023， 54（6）： 2365-2375.
	董亚洁，郝晓静，吴晋强，等. 基于绵羊角质形成细胞探究SHH通过Krox20调节IGFBP5的表达影响羊毛弯曲. 畜牧兽医学报， 2023， 54（6）： 2365-2375.
[10]	Yamamoto H， Flannery M L， Kupriyanov S， et al. Defective trophoblast function in mice with a targeted mutation of Ets2. Genes & Development， 1998， 12（9）： 1315-1326.
[11]	Yang H Y L， Yang Y L， Fang C H， et al. Fetal skin hair follicle development and morphology of Chinese Merino （Xinjiang Junken type）. Southwest China Journal of Agricultural Sciences， 2021， 34（1）： 202-207.
	杨涵羽璐，杨永林，方晨辉，等. 中国美利奴羊（新疆军垦型）胎儿期皮肤毛囊结构及形态学观察. 西南农业学报， 2021， 34（1）： 202-207.
[12]	Zhang Y J， Yin J， Li C Q， et al. Study on development of skin and hair follicle from fetal Inner Mongolian Arbas cashmere goats. Acta Veterinaria et Zootechnica Sinica， 2006， 37（8）： 761-768.
	张燕军，尹俊，李长青，等. 内蒙古阿尔巴斯绒山羊胎儿期皮肤毛囊发生发育规律研究. 畜牧兽医学报， 2006， 37（8）： 761-768.
[13]	Cloete E， Khumalo N P， Ngoepe M N. The what， why and how of curly hair： a review. Proceedings of the Royal Society A： Mathematical， Physical and Engineering Sciences， 2019， 475（2231）： DOI： 10.1098/rspa.2019.0516.
[14]	Li X L， Tao J Z， Ding W， et al. Analysis of wool traits and its relationship in Tan sheep during the er-mao period. China Animal Husbandry & Veterinary Medicine， 2019， 46（2）： 442-448.
	李向龙，陶金忠，丁伟，等. 滩羊二毛期羊毛性状分析及其相关性研究. 中国畜牧兽医， 2019， 46（2）： 442-448.
[15]	Yang H， Yang Y L， Liu S R， et al. Correlation analysis of the physical and chemical properties of wool among strains of Chinese Merino （Xinjiang Junken type）. China Animal Husbandry & Veterinary Medicine， 2011， 38（6）： 37-40.
	杨华，杨永林，刘守仁，等. 中国美利奴羊（新疆军垦型）品系间羊毛理化性能及性状相关分析. 中国畜牧兽医， 2011， 38（6）： 37-40.
[16]	Pickering N K， Blair H T， Hickson R E， et al. Genetic relationships between dagginess， breech bareness， and wool traits in New Zealand dual-purpose sheep. Journal of Animal Science， 2013， 91（10）： 4578-4588.
[17]	Chen Y， Fan Z M， Wang X X， et al. PI3K/Akt signaling pathway is essential for de novo hair follicle regeneration. Stem Cell Research & Therapy， 2020， 11（1）： 144.
[18]	Zhou H， Huang S N， Lv X Y， et al. Effect of CUX1 on the proliferation of Hu sheep dermal papilla cells and on the Wnt/β-catenin signaling pathway. Genes， 2023， 14（2）： 423.
[19]	Urban J， Qi L， Zhao H， et al. Comparison of hair manifestations in cardio-facio-cutaneous and costello syndromes highlights the influence of the RAS pathway on hair growth. Journal of the European Academy of Dermatology and Venereology， 2020， 34（3）： 601-607.
[20]	Wen F， Kumari N， Taylor Vi J G. RNA-seq and gene set enrichment analysis （GSEA） in peripheral blood mononuclear cells （PBMCs）. Methods in Molecular Biology， 2025， 2880： 179-192.
[21]	Spitz F， Furlong E E M. Transcription factors： from enhancer binding to developmental control. Nature Reviews Genetics， 2012， 13（9）： 613-626.
[22]	Christiano A M. Hair follicle epithelial stem cells get their sox on. Cell Stem Cell， 2008， 3（1）： 3-4.
[23]	Yan R Q. Research of FoxN1 on crimping fomation of wool. Taiyuan： Shanxi Agricultural University， 2019.
	闫瑞琴. FoxN1对羊毛弯曲形成作用的研究. 太原：山西农业大学， 2019.
[24]	Grisanti L， Clavel C， Cai X Q， et al. Tbx18 targets dermal condensates for labeling， isolation， and gene ablation during embryonic hair follicle formation. Journal of Investigative Dermatology， 2013， 133（2）： 344-353.
[25]	Liu R Q， Liu L D， Xu J D， et al. Identification of potential hub genes in alopecia areata. Experimental Dermatology， 2024， 33（10）： e70002.
[26]	Wang S H， Wu T Y， Sun J Y， et al. Single-cell transcriptomics reveals the molecular anatomy of sheep hair follicle heterogeneity and wool curvature. Frontiers in Cell and Developmental Biology， 2021， 9： 800157.
[27]	Lee H， Kim S Y， Kwon N， et al. Single-cell and spatial transcriptome analysis of dermal fibroblast development in perinatal mouse skin： dynamic lineage differentiation and key driver genes. Journal of Investigative Dermatology， 2024， 144（6）： 1238-1250.
[28]	Krieg P， Schuppler M， Koesters R， et al. Repetin （Rptn）， a new member of the “fused gene” subgroup within the S100 gene family encoding a murine epidermal differentiation protein. Genomics， 1997， 43（3）： 339-348.
[29]	Huber M， Siegenthaler G， Mirancea N， et al. Isolation and characterization of human repetin， a member of the fused gene family of the epidermal differentiation complex. Journal of Investigative Dermatology， 2005， 124（5）： 998-1007.
[30]	Pospiech E， Lee S D， Kukla-Bartoszek M， et al. Variation in the RPTN gene may facilitate straight hair formation in Europeans and East Asians. Journal of Dermatological Science， 2018， 91（3）： 331-334.
[31]	Zhao B R， Wu C L， Sammad A， et al. The fiber diameter traits of Tibetan cashmere goats are governed by the inherent differences in stress， hypoxic， and metabolic adaptations： an integrative study of proteome and transcriptome. BMC Genomics， 2022， 23（1）： 191.
[32]	Angwin C， Ghali N， van Dijk F S. Case report： Two individuals with AEBP1-related classical-like EDS： Further clinical characterisation and description of novel AEBP1 variants. Frontiers in Genetics， 2023， 14： 1148224.
[33]	Damak S， Su H， Jay N P， et al. Improved wool production in transgenic sheep expressing insulin-like growth factor 1. Nature Biotechnology， 1996， 14（2）： 185-188.
[34]	Jia B， Xi J F， Zhang S Y， et al. The developmental patterns of GH-r， IGF-1 and IGF-IR gene expression in sheep skin. Hereditas （Beijing）， 2006， 28（9）： 1078-1082.
[35]	Fu J Q， Zhang X Y， Wang D， et al. Analysis of the long non-coding and messenger RNA expression profiles in the skin tissue of super Merino and small-tailed Han sheep. Current Issues in Molecular Biology， 2024， 46（9）： 9588-9606.
[36]	Diao X G， Duan C H， Yao L Y， et al. Melatonin promotes the development of secondary hair follicles in adult cashmere goats by activating the Keap1-Nrf2 signaling pathway and inhibiting the inflammatory transcription factors NFkappaB and AP-1. International Journal of Molecular Sciences， 2023， 24（4）： 3403.
[37]	Jin M， Fan W Y， Lv S H， et al. LncRNA018392 promotes the proliferation of Liaoning cashmere goat skin fibroblasts by upregulating CSF1R through binding to SPI1. Molecular Biology Reports， 2024， 51（1）： 920.
[38]	Liu Y， Guerrero-Juarez C F， Xiao F， et al. Hedgehog signaling reprograms hair follicle niche fibroblasts to a hyper-activated state. Developmental Cell， 2022， 57（14）： 1758-1775.
[39]	Premanand A， Rajkumari B R. Bioinformatic analysis of gene expression data reveals Src family protein tyrosine kinases as key players in androgenetic alopecia. Frontiers in Medicine， 2023， 10： 1108358.
[40]	Ichikawa C， Izawa T， Juniantito V， et al. Rat hair follicle-constituting cells labeled by a newly-developed somatic stem cell-recognizing antibody： a possible marker of hair follicle development. Histology and Histopathology， 2013， 28（2）： 257-268.
[41]	Sedov E， Koren E， Chopra S， et al. THY1-mediated mechanisms converge to drive YAP activation in skin homeostasis and repair. Nature Cell Biology， 2022， 24（7）： 1049-1063.