基于RNA测序技术鉴定和分析绵羊毛囊发育相关的可变剪接事件

doi:10.3969/j.issn.1004-1524.20221409

浙江农业学报 ›› 2023, Vol. 35 ›› Issue (9): 2056-2067.DOI: 10.3969/j.issn.1004-1524.20221409

基于RNA测序技术鉴定和分析绵羊毛囊发育相关的可变剪接事件

袁晓春(), 王翊帆, 王雅艳, 孙昊然, 孟科, 李新海()

宁夏大学农学院,宁夏银川 750021

收稿日期:2022-09-28 出版日期:2023-09-25 发布日期:2023-10-09
作者简介:*李新海,E-mail:zhumei185@163.com
袁晓春(1997—),女,山东济南人,硕士研究生,研究方向为动物遗传育种与繁殖。E-mail:yuanxiaochun0206@163.com
通讯作者: 李新海
基金资助:
国家自然科学基金(31960650)

Identification and analysis of alternative splicing events related to sheep hair follicle development based on RNA sequencing technology

YUAN Xiaochun(), WANG Yifan, WANG Yayan, SUN Haoran, MENG Ke, LI Xinhai()

College of Agriculture, Ningxia University, Yinchuan 750021, China

Received:2022-09-28 Online:2023-09-25 Published:2023-10-09
Contact: LI Xinhai

摘要/Abstract

摘要：

可变剪接是调控动植物体内蛋白质多样性和遗传多样性的重要分子机制。为鉴定和分析杜泊羊毛囊发育过程中的可变剪接,分别于2019年9月(生长旺期,S1)、2020年1月(休止期,羊毛停止生长,S2)、2020年3月(生长初期,新羊毛生长导致旧羊毛脱落,S3)对杜泊成年母羊体侧中部皮肤组织取样,利用Illumina HiSeqTM 4000测序平台对样品进行转录组测序。利用rMATS软件鉴定样品中的可变剪接事件和差异剪接基因,在3组样品中共鉴定出128 033个可变剪接事件,其中外显子跳跃的比例最高,约占75.69%,内含子保留比例最少,约占2.03%。在S1vs S2、S1vs S3和S2vs S3中共筛选到3 448个显著(P<0.05)的差异剪接基因。GO(gene ontology)和KEGG(Kyoto Encyclopedia of Genes and Genomes)功能富集分析显示,差异剪接基因显著富集在血管内皮生长因子(VEGF)信号通路、丝裂原活化蛋白激酶(MAPK)信号通路、PI3K-Akt信号通路、Rap1(RAS-related protein 1)信号通路、Hippo信号通路、神经营养因子信号通路等与毛囊发育密切相关的通路上,表明筛选到的差异剪接基因在杜泊羊毛囊发育过程中发挥重要作用。并进一步筛选出可能参与绵羊毛囊发育相关的关键基因(SMAD2、FGFR2、BMP4、BCL2等)。本研究通过分析杜泊羊毛囊发育过程中的AS事件,揭示绵羊毛囊发育和羊毛呈周期性循环过程中相关遗传调控机制,研究结果可以为后续开展绵羊毛囊发育与羊毛生长呈周期性变化中发育相关基因功能分析与挖掘提供理论基础。

关键词: 绵羊, 可变剪接, 毛囊发育, 差异剪接基因

Abstract:

Alternative splicing is an important molecular mechanism that regulates protein diversity and genetic diversity in animals and plants. In order to identify and analyze the variable splicing during hair follicle development during depilation of Dorper sheep, the Illumina HiSeqTM 4000 sequencing platform was used to sequence the transcriptome of the skin tissue samples in the middle of the side of Dorper adult ewes in September 2019 (peak growth period, S1), January 2020 (telogen, wool growth stops, S2), and March 2020 (early growth period, new wool growth causes old wool to fall off, S3). Using the rMATS software to identify the variable splicing events and differential splicing genes in the samples, 128 033 variable splicing events were identified in three group samples, of which the proportion of skipped exons was the highest, accounting for 75.69%, and the proportion of retained introns was the lowest, accounting for 2.03%. A total of 3 448 significantly (P<0.05) different splicing genes were screened in S1 vs S2, S1 vs S3 and S2 vs S3. The functional enrichment analysis of GO (gene ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) showed that differential splicing genes were significantly enriched in VEGF (vascular endothelial growth factor) signal pathway, MAPK (mitogen-activated protein kinase) signal pathway, PI3K Akt signal pathway, Rap1 (RAS-related protein 1) signal pathway, Hippo signal pathway, neurotrophic factor signal pathway and other pathways closely related to hair follicle development, indicating that differential splicing genes played an important role in hair follicle development during depilation in Dorper sheep, The key genes (SMAD2, FGFR2, BMP4, BCL2, etc.) that may be involved in the hair follicle development of sheep depilation traits were further screened out. By analyzing the AS events in the hair follicle development process of Dorper sheep during depilation, this paper reveals the relevant genetic regulation mechanism in the process of hair follicle development and the periodic cycle of wool, which provides a theoretical basis for the subsequent analysis and excavation of the function of development related genes in the process of hair follicle development and the periodic change of wool growth during sheep depilation.

Key words: sheep, alternative splicing, hair follicle development, differentially spliced gene

中图分类号:

S826

袁晓春, 王翊帆, 王雅艳, 孙昊然, 孟科, 李新海. 基于RNA测序技术鉴定和分析绵羊毛囊发育相关的可变剪接事件[J]. 浙江农业学报, 2023, 35(9): 2056-2067.

YUAN Xiaochun, WANG Yifan, WANG Yayan, SUN Haoran, MENG Ke, LI Xinhai. Identification and analysis of alternative splicing events related to sheep hair follicle development based on RNA sequencing technology[J]. Acta Agriculturae Zhejiangensis, 2023, 35(9): 2056-2067.

图/表 8

参考文献 31

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基因Gene	正向引物Forward primer (5’→3’)	反向引物Reverse primer (5’→3’)	大小Size/bp
MT1C	CCCTCCTGCAAGAAGAGC	CACAGCAGCTGCACTTGTC	103
KRT23	TAGCAGATGGCAAGGTAAACAA	GTCAGATCATCCAGGGTCTTTC	160
S100A14	AGACCCTGATCAAGAATTTCCA	CCGAACTCCAGTTTAGAGTCGT	178
TPRXL	ATGCTCCCAGTGAAAGTACAGA	TCCATCTCACTGATAACTCAGCA	151
RPL6	GCCTCGGAAACTGTTGAGTC	GAGGCCCAGTCACAAGTAGC	173
GAPDH	TCCACGGCACAGTCAAGG	CACGCCCATCACAAACAT	239
RPL13	GTCAGGTACCACACGAAGGTT	CACCGAGATCCCAATGGTC	105

基因Gene	正向引物Forward primer (5’→3’)	反向引物Reverse primer (5’→3’)	大小Size/bp
MT1C	CCCTCCTGCAAGAAGAGC	CACAGCAGCTGCACTTGTC	103
KRT23	TAGCAGATGGCAAGGTAAACAA	GTCAGATCATCCAGGGTCTTTC	160
S100A14	AGACCCTGATCAAGAATTTCCA	CCGAACTCCAGTTTAGAGTCGT	178
TPRXL	ATGCTCCCAGTGAAAGTACAGA	TCCATCTCACTGATAACTCAGCA	151
RPL6	GCCTCGGAAACTGTTGAGTC	GAGGCCCAGTCACAAGTAGC	173
GAPDH	TCCACGGCACAGTCAAGG	CACGCCCATCACAAACAT	239
RPL13	GTCAGGTACCACACGAAGGTT	CACCGAGATCCCAATGGTC	105

样品名 Sample name	原始读段 Raw reads	干净读段 Clean reads	Q30/%	GC含量 GC content/%	唯一对比到的read数占比 Unique_mapped reads ratio/%	对比率 Total_mapped ratio/%
S1-1	80 305 380	80 151 468	92.90	48.51	85.82	93.65
S1-2	88 681 572	88 514 636	93.52	50.33	84.78	96.29
S1-3	88 639 604	88 435 920	93.76	50.48	84.18	95.94
S1-4	82 302 900	82 082 050	93.75	52.49	80.81	96.52
S1-5	95 132 446	94 861 532	93.53	50.14	83.97	95.97
S2-1	101 606 896	101 305 036	93.08	49.02	87.06	95.82
S2-2	111 482 124	111 138 096	93.53	50.04	85.72	96.13
S2-3	78 132 386	77 918 790	94.08	49.78	86.16	96.70
S2-4	88 934 292	88 662 238	93.50	51.37	81.77	94.38
S2-5	88 841 684	88 527 154	93.48	50.42	85.05	95.57
S3-1	94 155 288	93 804 848	92.47	51.43	83.88	95.15
S3-2	100 794 688	100 434 556	93.45	50.76	82.96	94.34
S3-3	79 139 424	78 899 488	94.13	51.69	80.02	95.91
S3-4	79 187 140	78 889 182	93.51	53.35	82.35	94.68
S3-5	80 678 998	80 361 468	93.72	51.84	77.34	92.41

样品名 Sample name	原始读段 Raw reads	干净读段 Clean reads	Q30/%	GC含量 GC content/%	唯一对比到的read数占比 Unique_mapped reads ratio/%	对比率 Total_mapped ratio/%
S1-1	80 305 380	80 151 468	92.90	48.51	85.82	93.65
S1-2	88 681 572	88 514 636	93.52	50.33	84.78	96.29
S1-3	88 639 604	88 435 920	93.76	50.48	84.18	95.94
S1-4	82 302 900	82 082 050	93.75	52.49	80.81	96.52
S1-5	95 132 446	94 861 532	93.53	50.14	83.97	95.97
S2-1	101 606 896	101 305 036	93.08	49.02	87.06	95.82
S2-2	111 482 124	111 138 096	93.53	50.04	85.72	96.13
S2-3	78 132 386	77 918 790	94.08	49.78	86.16	96.70
S2-4	88 934 292	88 662 238	93.50	51.37	81.77	94.38
S2-5	88 841 684	88 527 154	93.48	50.42	85.05	95.57
S3-1	94 155 288	93 804 848	92.47	51.43	83.88	95.15
S3-2	100 794 688	100 434 556	93.45	50.76	82.96	94.34
S3-3	79 139 424	78 899 488	94.13	51.69	80.02	95.91
S3-4	79 187 140	78 889 182	93.51	53.35	82.35	94.68
S3-5	80 678 998	80 361 468	93.72	51.84	77.34	92.41

基于RNA测序技术鉴定和分析绵羊毛囊发育相关的可变剪接事件

Identification and analysis of alternative splicing events related to sheep hair follicle development based on RNA sequencing technology

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