基于转录组测序研究调控黄羽肉鸡干毛性状关键基因和信号通路

doi:10.3864/j.issn.0578-1752.2024.01.014

中国农业科学 ›› 2024, Vol. 57 ›› Issue (1): 204-215.doi: 10.3864/j.issn.0578-1752.2024.01.014

基于转录组测序研究调控黄羽肉鸡干毛性状关键基因和信号通路

姬改革¹(), 陈智武²(), 单艳菊¹, 刘一帆¹, 屠云洁¹, 邹剑敏¹, 章明¹, 巨晓军¹, 束婧婷¹(), 张海涛³, 唐燕飞⁴, 蒋华莲⁴

¹ 江苏省家禽科学研究所/江苏省家禽遗传育种重点实验室，江苏扬州 225125
² 广西金陵农牧集团有限公司，南宁 530000
³ 江苏立华牧业股份有限公司，江苏常州 213168
⁴ 广西富凤农牧集团有限公司，南宁 530024

收稿日期:2023-05-09 接受日期:2023-10-31 出版日期:2024-01-01 发布日期:2024-01-10
通信作者:
束婧婷，E-mail：shujingting@163.com
联系方式: 姬改革，E-mail：jigaige@126.com。陈智武，E-mail：ubtech@163.com。姬改革与陈智武为同等贡献者。
基金资助:
江苏省自然科学基金(BK20210955); 江苏省种业振兴揭榜挂帅项目(JBGS〔2021〕107); 江苏现代农业（肉鸡）产业技术体系(JATS[2022]399); 江苏省农业重大新品种创制项目(PZCZ201728); 现代农业产业技术体系建设专项资金(CARS-41)

Study of Key Genes and Signaling Pathways Regulating Dry Feather Traits in Yellow-Feathered Broiler Chickens Based on Transcriptome Analysis

JI GaiGe¹(), CHEN ZhiWu²(), SHAN YanJu¹, LIU YiFan¹, TU YunJie¹, ZOU JianMin¹, ZHANG Ming¹, JU XiaoJun¹, SHU JingTing¹(), ZHANG HaiTao³, TANG YanFei⁴, JIANG HuaLian⁴

¹ Key Laboratory of Poultry Genetics and Breeding of Jiangsu Province/Jiangsu Institute of Poultry Sciences, Yangzhou 225125, Jiangsu
² Guangxi Jinling Agriculture and Animal Husbandry Group Co., Ltd., Nanning 530000
³ Jiangsu Lihua Animal Husbandry Stock Co., Ltd., Changzhou 213168, Jiangsu
⁴ Guangxi Fufeng Farm Group Co., Ltd., Nanning 530024

Received:2023-05-09 Accepted:2023-10-31 Published:2024-01-01 Online:2024-01-10

摘要/Abstract

摘要：

【目的】通过比较干毛和未干毛羽毛毛囊的形态学和基因表达差异，挖掘调控黄羽肉鸡羽毛干毛性状的重要候选基因和信号通路。【方法】分别采集背部未干毛和干毛羽毛皮肤组织样本各3个，运用组织切片技术，比较干毛和未干毛羽毛毛囊形态学差异；运用RNA-seq技术，比较两组样本之间的差异表达基因（differentially expressed genes，DEGs），并对其进行功能富集分析和蛋白互作网络构建；采用荧光定量PCR技术（RT-qPCR）对测序结果准确性进行验证。【结果】证实未干毛羽毛的毛囊处于生长期，已干毛的羽毛毛囊处于静止期。以未干毛毛囊（生长期）皮肤样本为对照，在干毛毛囊（静止期）皮肤样本中发现了942个DEGs(|fold-change|>2和P<0.05)，其中384个基因表达下调，558个基因表达上调。Go功能分析显示细胞分裂、周期调控等相关生物过程被显著富集(P<0.05)。KEGG分析发现，MAPK、TGF-β、p53及DNA复制等相关信号通路被显著富集(P<0.05)。构建差异蛋白相互作用（PPI）网络，通过CytoHubba分析获得6个hub基因，分别为CDK1、MAD2L1、BUB1、CCNB2、PLK1和BUB1B。GSEA富集分析筛选到紧密连接、胰岛素、MAPK、TGF-β和细胞周期等信号通路与鸡羽毛毛囊生长周期显著关联（|NES|>1, FDR<0.25）。RT-qPCR结果显示8个DEGs表达趋势与RNA-seq结果基本一致。【结论】鸡羽毛的干毛性状与毛囊周期发育相关，MAPK和TGF-β等信号通路可能通过调控细胞周期相关基因的表达在羽毛生长发育中发挥重要作用；结果为进一步深入了解黄羽肉鸡羽毛干毛性状分子调控机制提供了基础资料。

关键词: 鸡, 干毛, 毛囊, RNA-seq, 基因, 信号通路

Abstract:

【Objective】 The study aimed to identify important candidate genes and signaling pathways associated with dry feather traits by comparing the morphological and gene expression differences between the feather follicles of dry and undried feathers. 【Method】 Three samples of skin tissue were selected from each of the undried and dried feathers. The histological examinations were used to compare the morphological differences between the feather follicles of dried and undried feathers. RNA-seq technology was employed to compare the differentially expressed genes (DEGs) between the two groups of skin samples. The accuracy of transcriptome results was validated by using the fluorescent quantitative PCR technique (RT-qPCR). 【Result】 By histological H.E staining, it was confirmed that the feather follicles of the undried feathers were in the growth phase, while the feather follicles of the dried feathers were in the resting phase. The feather follicle skin samples at the growth stage were used as controls, 942 DEGs were identified in resting feather follicle samples (|fold-change|>2 and P<0.05), including 384 significantly down-regulated DEGs and 558 upregulated DEGs. Gene ontology (Go) analysis suggested that the DEGs were significantly enriched in cell division, cycle regulation, and other related biological processes. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway results showed that the DEGs were related to MAPK, TGF-β, p53, and cell cycle-related signaling pathways. Protein-protein interaction (PPI) network was constructed and six hub genes were obtained by CytoHubba analysis, including CDK1, MAD2L1, BUB1, CCNB2, PLK1, and BUB1B. Gene Set Enrichment Analysis (GSEA) results indicated that the signaling pathways related to the tight junction, insulin, MAPK, TGF-β, and cell cycle-related pathways were significantly associated with the growth cycle of chicken feather follicles. The expression patterns of 8 DEGs detected by RT-qPCR were consistent with the RNA-seq results. 【Conclusion】 In summary, the dry feather traits of chickens were related to the development of feather follicle cycles. Signaling pathways such as MAPK and TGF-β might play important roles in feather growth and development by regulating the expression of cell cycle-related genes. The study provided clues for understanding the molecular regulatory mechanisms for dry feather traits in yellow-feathered broiler chickens.

Key words: chicken, dry feather, feather follicle, RNA-seq, gene, signaling pathway

姬改革, 陈智武, 单艳菊, 刘一帆, 屠云洁, 邹剑敏, 章明, 巨晓军, 束婧婷, 张海涛, 唐燕飞, 蒋华莲. 基于转录组测序研究调控黄羽肉鸡干毛性状关键基因和信号通路[J]. 中国农业科学, 2024, 57(1): 204-215.

JI GaiGe, CHEN ZhiWu, SHAN YanJu, LIU YiFan, TU YunJie, ZOU JianMin, ZHANG Ming, JU XiaoJun, SHU JingTing, ZHANG HaiTao, TANG YanFei, JIANG HuaLian. Study of Key Genes and Signaling Pathways Regulating Dry Feather Traits in Yellow-Feathered Broiler Chickens Based on Transcriptome Analysis[J]. Scientia Agricultura Sinica, 2024, 57(1): 204-215.

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基因 Gene symbol	序列号 Accession number	引物序列 Primer sequence (5′→3′)	产物长度 Product length (bp)
β-actin	NM_205518.2	F: GCTGAACTCCATCTTGGTTCCC R: TGCTTCTAGGGTCACTCGCA	150
BUB1	NM_001012870.1	F: GCTGAACTCCATCTTGGTTCCC R: TGCTTCTAGGGTCACTCGCA	112
BIRC5	NM_001012319.1	F:CTGGATAAAAAGCGGACCAA R: AACCTAAGGGCCCATGTTCT	246
CDK1	NM_205314.1	F:GGAACGCATGTCCAAAACTT R: GCAGGCAGGCAAAGATAAAG	236
NEK2	NM_001031050.2	F:ATGTCTTCCTGGATGGCAAG R: TCTGCCAACTCCTTCTGGTT	241
CDC6	XM_040691823.1	F:GTGTCCTCTCGGAGGTGTTT R: AGGCACTCTGTCTGGTCCAC	214
ANXA1	NM_206906.1	F:AAAAACTCCACCTGGCAATG R: CCACATAAAGCGACCAGGAT	193
BCL2	NM_205339.2	F: TGACACCTCGATCTCACAGG R: CATGCAACACACTGGGATTC	247
EDA2R	NM_001083360.1	F: ACCCTCATCAACCGCATCCA R: GCCTTGCGGTAAAACCCTGG	89
FZD4	NM_204099.1	F:AATGTCACCAAGATGCCCAACC R:AGACCGAACAAAGGAAGAACTGGA	130

样品 Sample	原始序列 Raw reads	有效序列 Clean reads	Q30 (%)	GC (%)	比对率 Mapped on reference	未比对率 Unmapped	唯一位置比对 Uniquely mapped
H1-1	40968048	38142462	92.33	48.605	36255024 (95.05%)	1887438 (4.95%)	35196169 (92.28%)
H1-2	46296028	43781388	93.07	49.900	41547151 (94.9%)	2234237 (5.1%)	40274977 (91.99%)
H1-3	48737026	45301826	92.01	48.995	42668140 (94.19%)	2633686 (5.81%)	41786822 (92.24%)
H2-1	46250350	43867410	93.33	49.985	41559772 (94.74%)	2307638 (5.26%)	40597907 (92.55%)
H2-2	43917200	40983494	92.28	49.945	38739383 (94.52%)	2244111 (5.48%)	37804671 (92.24%)
H2-3	42372910	39985456	93.17	49.750	38040749 (95.14%)	1944707 (4.86%)	36991013 (92.51%)

基因 Gene	MCC	基因 Gene	Degree	基因 Gene	MNC	基因 Gene	EPC
CDK1	5.11E+19	CDK1	56	CDK1	55	APITD1	85.564
BUB1	5.11E+19	PLK1	46	PLK1	46	AURKA	85.564
PLK1	5.11E+19	CCNB2	44	CCNB2	43	CDK1	85.564
CCNB2	5.11E+19	CENPE	43	CENPE	43	TPX2	85.564
NDC80	5.11E+19	BUB1	41	BUB1	41	BIRC5	85.564
CENPE	5.11E+19	NDC80	41	NDC80	41	MAD2L1	85.564
BUB1B	5.11E+19	BUB1B	37	BUB1B	37	BUB1	85.564
MAD2L1	5.11E+19	MAD2L1	34	KIF11	34	CCNB2	85.564
CENPF	5.11E+19	KIF11	34	MAD2L1	33	PLK1	85.564
INCENP	5.11E+19	AURKA	32	AURKA	31	BUB1B	85.564

基于转录组测序研究调控黄羽肉鸡干毛性状关键基因和信号通路

Study of Key Genes and Signaling Pathways Regulating Dry Feather Traits in Yellow-Feathered Broiler Chickens Based on Transcriptome Analysis

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