基于转录组学分析受外源褪黑素诱导调控羊绒生长的mRNA和lncRNA的可变剪接

doi:10.16431/j.cnki.1671-7236.2025.07.018

摘要/Abstract

摘要： 【目的】分析转录后水平绒山羊皮肤中被外源褪黑素诱导参与调控绒生长发生可变剪接(alternative splicing,AS)的mRNA和长链非编码RNA(long non-coding RNA,lncRNA)，为探究AS调节对褪黑素促山羊绒生长的影响提供参考。【方法】选取6只罕山白绒山羊，随机分为2组：对照组和埋植组，每组3个重复。以自然年为试验周期，每月采集皮肤样本进行转录组测序，使用Bowtie、TopHat、Cufflinks、Cuffmerge、Cuffdiff、Cuffcompare、CPAT和CPC软件分析差异表达mRNA(differentially expressed mRNA,DE-mRNA)和差异表达lncRNA (differentially expressed lncRNA,DE-lncRNA)，使用rMATS软件识别绒山羊皮肤转录组mRNA和lncRNA差异可变剪接事件(differential alternative splicing events,DASE)。【结果】筛选到1～12月组间DE-mRNA(FDR≤0.05)数目分别为23、93、43、652、1 178、194、353、357、435、36、55和52个，DE-lncRNA(FDR≤0.05)数目分别为15、20、10、40、191、50、63、33、79、7、9和5个。mRNA和lncRNA发生组间DASE共715和569个；外显子跳跃(skipping exon,SE)和互斥外显子(mutually exclusive exons,MXE)为mRNA最主要的AS方式，SE、内含子保留(retained intron，RI)和3'-可变剪接位点(alternative 3'-splice site,A3SS)为lncRNA最主要的AS方式。染色体上mRNA的DASE数目排名前三为Chr1、Chr5和Chr10/Chr11(并列)，lncRNA的DASE数目排名前三为Chr1、Chr19和Chr10。绒山羊皮肤中许多mRNA和lncRNA存在2个或多个剪接异构体，确定了443个DAS-mRNA和271个DAS-lncRNA，DAS-mRNA数目排名前三为10、6和5月，DAS-lncRNA数目排名前三为5、4和8月。DE-mRNA和DE-lncRNA中分别有67和7个发生了DASE。在外源褪黑素诱导下，绒山羊皮肤中发生AS的mRNA和lncRNA与皮尔逊相关系数(Pearson’s correlation coefficient，PCC)排名前十(PCC＞0.800)的334个DE-mRNA和8个DE-lncRNA组成AS调控网络。【结论】绒山羊皮肤中存在丰富的ASE，除SE外，mRNA偏爱MXE，而lncRNA偏爱RI的AS方式；与mRNA相比，lncRNA的DASE在染色体上的分布具有特异性。受外源褪黑素诱导，山羊绒生长旺盛期和生长前期是mRNA发生AS的主要时期，而山羊绒生长前期和生长期是lncRNA发生AS的主要时期。试验构建了发生AS的DE-mRNA和DE-lncRNA在绒山羊皮肤转录组的潜在互作网络，DE-mRNA和DE-lncRNA在转录后水平通过AS方式参与调控山羊绒生长，为进一步研究AS调节对褪黑素促进山羊绒生长的影响提供参考。

关键词: 绒山羊; 皮肤; 褪黑素; 转录组测序; 长链非编码RNA; 可变剪接

Abstract: 【Objective】 This study was aimed to analyze mRNA and long non-coding RNA (lncRNA) in cashmere goat skin induced by exogenous melatonin to participate in the regulation of alternative splicing (AS) of cashmere growth at post transcriptional level,so as to provide a reference for exploring the effect of AS regulation on melatonin promoting cashmere growth in goats. 【Method】 Six Hanshan White cashmere goats were selected and randomly assigned to control and implant groups with three replicates in each group.Take the natural year as the experiment period,skin samples were collected monthly for RNA-Seq analysis.UsedBowtie,TopHat,Cufflinks,Cuffmerge,Cuffdiff,Cuffcompare,CPAT and CPC software were used to analyze differentially expressed mRNA (DE-mRNA) and differentially expressed lncRNA (DE-lncRNA),with rMATS software to identify differential alternative splicing events (DASE) of mRNA and lncRNA in cashmere goat skin transcriptome. 【Result】 The number of DE-mRNA (FDR≤0.05) between the two groups from January to December was 23,93,43,652,1 178,194,353,357,435,36,55 and 52,respectively.The number of DE-lncRNA (FDR≤0.05) between the two groups from January to December was 15,20,10,40,191,50,63,33,79,7,9 and 5,respectively.The number of DASE between the two groups involving mRNA and lncRNA was 715 and 569,respectively.Skipping exon (SE) and mutually exclusive exons (MXE) were the main alternative splicing ways of mRNA.SE,retained intron (RI) and alternative 3'-splice site (A3SS) were the main AS ways of lncRNA.The top 3 chromosomes of DASE number in mRNA were Chr1,Chr5 and Chr10/Chr11 (juxtaposition),and the top 3 chromosomes of DASE in lncRNA were Chr1,Chr19 and Chr10,respectively.Two or more splicing isoforms of mRNA and lncRNA were found in cashmere goat skin.A total of 443 DAS-mRNA and 271 DAS-lncRNA were identified.The top 3 months of DAS-mRNA distribution were October,June and May,and the top 3 months of DAS-lncRNA distribution were May，April and August,respectively.A total of 67 DE-mRNA and 7 DE-lncRNA underwent DASE.The AS regulatory network induced by exogenous melatonin，which composed of 334 DE-mRNA and 8 DE-lncRNA associated with mRNA and lncRNA underwent AS of Pearson’s correlation coefficient (PCC) ranking in top 10 (PCC＞0.800). 【Conclusion】 There were abundant ASE in cashmere goat skin,except for SE,mRNA preferred the alternative splicing way of MXE,while lncRNA preferred RI.Compared with mRNA,the distribution of DASE in lncRNA on chromosomes was specific.When induced by exogenous melatonin,the cashmere fast-growing and the early growth period were main periods for mRNA,while the early growth and the growth period were the main periods for lncRNA occurrence of AS.The potential interaction network between DE-mRNA and DE-lncRNA involved in the transcriptome of cashmere goat skin undergoing AS was constructed,DE-mRNA and DE-lncRNA participated in regulating cashmere growth through AS at the post transcriptional level,providing a reference for further exploring the effect of AS regulation on melatonin promoting cashmere growth.

Key words: cashmere goat; skin; melatonin; RNA-Seq; lncRNA; AS

中图分类号:

S813.3

贾纯琰, 孙燕勇, 包永红, 张文广, 杜晨光. 基于转录组学分析受外源褪黑素诱导调控羊绒生长的mRNA和lncRNA的可变剪接[J]. 中国畜牧兽医, 2025, 52(7): 3165-3177.

JIA Chunyan, SUN Yanyong, BAO Yonghong, ZHANG Wenguang, DU Chenguang. Alternative Splicing Analysis of mRNA and lncRNA Induced by Exogenous Melatonin for Regulating Cashmere Growth Based on Transcriptomics[J]. China Animal Husbandry & Veterinary Medicine, 2025, 52(7): 3165-3177.

参考文献

[1] SU T,HOLLAS M A R,FELLERS R T,et al.Identification of splice variants and isoforms in transcriptomics and proteomics[J].Annual Review of Biomedical Data Science,2023,6:357-376.
[2] WANG E T,SANDBERG R,LUO S,et al.Alternative isoform regulation in human tissue transcriptomes[J].Nature,2008,456(7221):470-476.
[3] KIEGLE E A,ALEX G,ELIA L,et al.A genomic view of alternative splicing of long non-coding RNAs during rice seed development reveals extensive splicing and lncRNA gene families[J].Frontiers in Plant Science,2018,9:115.
[4] 岳春旺.褪黑激素调控绒山羊绒毛生长机理的研究[D].北京:中国农业大学,2007.YUE C W.Research on the mechanism of melatonin regulating cashmere growth in cashmere goat[D].Beijing:China Agricultural University,2007.(in Chinese)
[5] YE G,XIAO L W,HAI L Y,et al.Comparative transcriptome analysis of fetal skin reveals key genes related to hair follicle morphogenesis in cashmere goats[J].PLoS One,2016,11(3):e0151118.
[6] 季小阳,刘斌,白雪，等.绒山羊次级毛囊基因表达滞后性的转录组分析[J].中国畜牧兽医,2016,43(7):1694-1701.JI X Y,LIU B,BAI X,et al.Transcriptome survey reveals hysteresis in secondary hair follicle for gene regulation in cashmere goat[J].China Animal Husbandry & Veterinary Medicine,2016,43(7):1694-1701.(in Chinese)
[7] FENG Y,ZHI H L,MENG Z,et al.Skin transcriptome reveals the periodic changes in genes underlying cashmere (ground hair) follicle transition in cashmere goats[J].BMC Genomics,2020,21(1):392.
[8] HUI T Y,ZHENG Y Y,YUE C,et al.Screening of cashmere fineness-related genes and their ceRNA network construction in cashmere goats[J].Scientific Reports,2021,11(1):21977.
[9] YUAN G,WEI G S,FEI H,et al.Effect of fibroblast growth factor 10 and an interacting non-coding RNA on secondary hair follicle dermal papilla cells in cashmere goats’ follicle development assessed by whole-transcriptome sequencing technology[J].Animals (Basel),2023,13(13):2234.
[10] 贾纯琰,付绍印,丽春,等.外源褪黑素诱导下Hippo信号通路调控山羊绒生长mRNA和lncRNA的生物信息学分析[J].中国农业大学学报,2023,28(5):128-139.JIA C Y,FU S Y,LI C,et al.Bioinformatics analysis of mRNA and lncRNA induced by exogenous melatonin that regulated cashmere growth by Hippo signaling pathway[J].Journal of China Agricultural University,2023,28(5):128-139.(in Chinese)
[11] 陆庆伟,唐森,李彬,等.基于转录组测序筛选疆南绒山羊次级毛囊周期发育相关的circRNA[J].中国畜牧兽医,2024,51(5):1793-1806.LU Q W,TANG S,LI B,et al.Screening of circRNA related to secondary hair follicle cycle development in Jiangnan cashmere goats based on transcriptome sequencing[J].China Animal Husbandry & Veterinary Medicine,2024,51(5):1793-1806.(in Chinese)
[12] LELE W,YAN J Z,MENG Z,et al.SNP discovery from transcriptome of cashmere goat skin[J].Asian-Australasian Journal of Animal Sciences,2015,28(9):1235-1243.
[13] FAN Y X,WU R B,QIAO X,et al.Hair follicle transcriptome profiles during the transition from anagen to catagen in cashmere goat (Capra hircus)[J].Genetics and Molecular Research,2015,14(4):17904-17915.
[14] MIN Y,SHEN S,KUN Z D,et al.Skin transcriptome reveals the intrinsic molecular mechanisms underlying hair follicle cycling in cashmere goats under natural and shortened photoperiod conditions[J].Scientific Reports,2017,7(1):13502.
[15] WANG S,LI F,LIU J,et al.Integrative analysis of methylome and transcriptome reveals the regulatory mechanisms of hair follicle morphogenesis in cashmere goat[J].Cells,2020,9(4):969.
[16] XIAO Y J,JIAN X W,BIN L,et al.Comparative transcriptome analysis reveals that a ubiquitin-mediated proteolysis pathway is important for primary and secondary hair follicle development in cashmere goats[J].PLoS One,2016,11(10):e0156124.
[17] ZHANG Y J,WANG L L,LI Z,et al.Transcriptome profiling reveals transcriptional and alternative splicing regulation in the early embryonic development of hair follicles in the cashmere goat[J].Scientific Reports,2019,9(1):17735.
[18] ZHANG Y L,LI F,SHI Y J,et al.Comprehensive transcriptome analysis of hair follicle morphogenesis reveals that lncRNA-H19 promotes dermal papilla cell proliferation through the chi-miR-214-3p/β-catenin axis in cashmere goats[J].International Journal of Molecular Sciences,2022,23(17):10006.
[19] 贾纯琰,孙燕勇,包永红,等.调控山羊绒生长mRNA和lncRNA基因簇及关键信号通路的生物信息学分析[J].中国畜牧兽医,2023,50(11):4311-4324.JIA C Y,SUN Y Y,BAO Y H,et al.Bioinformatics analysis of mRNA and lncRNA gene clusters and key signal pathways regulating cashmere growth[J].China Animal Husbandry & Veterinary Medicine,2023,50(11):4311-4324.(in Chinese)
[20] 赵濛,刘志红,奈日乐,等.基于高通量转录组测序阿尔巴斯型绒山羊绒毛生长周期差异表达基因分析[J].中国农业大学学报,2019,24(2):69-81.ZHAO M,LIU Z H,NAI R L,et al.Study on differentially expressed genes in the cycle of follicle growth of Arbas cashmere goat based on transcriptomes with high-throughput RNA-Seq technology[J].Journal of China Agricultural University,2019,24(2):69-81.(in Chinese)
[21] ZHANG Y,WU K,WANG L,et al.Comparative study on seasonal hair follicle cycling by analysis of the transcriptomes from cashmere and milk goats[J].Genomics,2020,112(1):332-345.
[22] 李莹莹,董华娇,梁千千,等.基于RNA测序技术鉴定和分析绒山羊毛囊不同生长期mRNA的可变剪接[J].中国草食动物科学,2024,44(4):1-7.LI Y Y,DONG H J,LIANG Q Q,et al.Identification and analysis of alternative splicing of mRNA related to different growth stages of cashmere goat hair follicles based on RNA sequencing technology[J].China Herbivore Science,2024,44(4):1-7.(in Chinese)
[23] 岳春旺,张微,孔祥浩,等.褪黑激素对内蒙古白绒山羊产绒性能的影响[J].中国畜牧杂志,2007,7:32-34.YUE C W,ZHANG W,KONG X H,et al.Effect of melatonin on cashmere performance in Inner Mongolia White cashmere goats[J].Chinese Journal of Animal Science,2007,7:32-34.(in Chinese)
[24] TRAPNELL C,PACHTER L,SALZBERG S L.Tophat:Discovering splice junctions with RNA-Seq[J].Bioinformatics,2009,25:1105-1111.
[25] TRAPNELL C,ROBERTS A,GOFF L,et al.Differential gene and transcript expression analysis of RNA-Seq experiments with TopHat and Cufflinks[J].Nature Protocols,2012,7(3):562-578.
[26] GHOSH S,CHAN C K K.Analysis of RNA-Seq data using Tophat and Cufflinks[J].Plant Bioinformatics,2016,1347:339-361.
[27] PARK J W,TOKHEIM C,SHEN S,et al.Identifying differential alternative splicing events from RNA sequencing data using RNA-Seq-MATS[J].Methods in Molecular Biology,2013,1038:171-179.
[28] SHANNON P,MARKIEL A,OZIER O,et al.Cytoscape:A software environment for integrated models of biomolecular interaction networks[J].Genome Research,2003,13(11):2498-2504.
[29] ROY B,HAUPT L M,GRIFFITHS L R.Review:Alternative splicing (AS) of genes as an approach for generating protein complexity[J].Current Genomics,2013,14(3):182-194.
[30] 李颖,宋峰,苏德其其格,等.绒山羊皮肤全长转录组测序及生物信息学分析[J].中国农业大学学报,2022,27(12):170-179.LI Y,SONG F,SU D,et al.Sequencing and bioinformatics analysis of the full-length transcriptome of cashmere goat skin[J].Journal of China Agricultural University,2022,27(12):170-179.(in Chinese)
[31] WODARZ A,NUSSE R.Mechanisms of Wnt signaling in development[J].Annual Review of Cell and Developmental Biology,1998,14(1):59-88.
[32] GALLO V,CIRILLO E,GIARDINO G,et al.FOXN1 deficiency:From the discovery to novel therapeutic approaches[J].Journal of Clinical Immunology,2017,37(8):751-758.
[33] 贾纯琰.受外源褪黑素诱导的绒山羊皮肤中调控绒生长的mRNA和lncRNA的研究[D].呼和浩特:内蒙古农业大学,2022.JIA C Y.mRNA and lncRNA induced by exogenous melatonin that regulate cashmere growth in cashmere goat skin[D].Hohhot:Inner Mongolia Agricultural University,2022.(in Chinese)
[34] MÜLLER R S,HANDJISKI B,VEEN C,et al.A comprehensive guide for the accurate classication of murine hair follicles in distinct hair cycle stages[J].The Journal of Investigative Dermatology,2001,117(1):1-15.
[35] GE W,WANG S H,SUN B,et al.Melatonin promotes cashmere goat (Capra hircus) secondary hair follicle growth:A view from integrated analysis of long non-coding and coding RNAs[J].Cell Cycle,2018,17(10):1255-1267.
[36] CHEN Y,FAN Z,WANG X,et al.PI3K/Akt signaling pathway is essential for de novo hair follicle regeneration[J].Stem Cell Research & Therapy,2020,11:144.
[37] TOBIN D J,FOITZIK K,REINHECKEL T,et al.The lysosomal protease cathepsin L is an important regulator of keratinocyte and melanocyte differentiation during hair follicle morphogenesis and cycling[J].American Journal of Pathology,2002,160(5):1807-1821.
[38] SEREZANI A P,BOZDOGAN G,SEHRA S,et al.IL-4 impairs wound healing potential in the skin by repressing fibronectin expression[J].Journal of Allergy & Clinical Immunology,2016,139(1):142-151.
[39] TRÜEB R M.Hormones and hair growth[J].Der Hautarzt,2010,61(6):487-495.