绵羊不同发育阶段背最长肌组织中可变剪接的鉴定与分析

doi:10.13560/j.cnki.biotech.bull.1985.2018-1078

摘要/Abstract

摘要： 可变剪接（Alternative splicing,AS）是动植物体内蛋白质多样性和遗传多样性的重要调控机制。为鉴定和分析绵羊不同发育阶段背最长肌组织中可变剪接,对多浪绵羊妊娠90日龄胎儿（F90）、出生后30日龄羔羊（L30）和成年3岁羊（A3Y）的背最长肌组织进行转录组测序,利用rMATS软件鉴定样品中的可变剪接事件和差异剪接基因（Differential splicing gene,DSG）,并对DSG进行GO和KEGG功能富集分析。结果表明,绵羊背最长肌组织在F90、L30和A3Y时期分别鉴定出13 923、11 959和12 164个可变剪接事件,其中外显子跳跃（Skipped exons,SE）的比例最高,约为70.69%,5'端可变剪接位点（Alternative 5' splice sites,A5SS）、3'端可变剪接位点（Alternative 3'splice sites,A3SS）、互斥外显子（Mutually exclusive exons,MXE）和内含子保留（Retained introns,RI）的比例分别约为7.28%、11.18%、5.96%和4.84%。在F90_vs_L30、F90_vs_A3Y和L30_vs_A3Y比较组中分别鉴定到 2 545、2 689和1 701个显著的差异剪接基因（P<0.05）。GO和KEGG功能富集分析显示,差异剪接基因显著富集到横纹肌发育、肌肉结构发育、肌细胞分化、肌膜、胰岛素信号通路、Wnt信号通路、MAPK信号通路等与肌肉发育密切相关的通路上。上述结果表明可变剪接在背最长肌发育中发挥重要作用。

关键词: 绵羊, 可变剪接, 差异剪接基因, RNA-seq

Abstract: The alternative splicing（AS）is an important regulatory mechanism of protein diversity and genetic diversity in animals and plants.The objective of this study was to identify and analyze alternative splicing in the longissimus dorsi of sheep at different developmental stages. The transcriptomic profiling of the longissimus dorsi of 90-day-old fetus（F90）,30-day-old lamb（L30）and adult 3-year-old sheep（A3Y）were sequenced,and the alternative splicing event（AS）and differential splicing gene（DSG）were identified using rMATS software. The GO function enrichment and KEGG pathway analysis were performed for DSG. The results showed that 13 923,11 959 and 12 164 alternative splicing events were identified in genome of longissimus dorsi tissues in the F90,L30 and A3Y,respectively,and the proportion of skipped exons（SE）was the highest,about 70.69% of the total number of AS. The proportion of 5' splice sites（A5SS）,3'splice sites（A3SS）,mutually exclusive exons（MXE）and retained introns（RI）was about 7.28%,11.18%,5.96%,and 4.84%,respectively. 2 545,2 689,and 1 701 significant differentially spliced genes（P<0.05）were identified in the F90_vs_L30,F90_vs_A3Y and L30_vs_A3Y groups,respectively（P < 0.05）. The GO and KEGG analysis showed that differentially spliced genes were significantly enriched in striated muscle development,muscle structure development,myocyte differentiation,sarcolemma,insulin signaling pathway,Wnt signaling pathway,MAPK signaling pathway and other pathways closely related to muscle development. These results suggest that alternative splicing plays an important role in the development of the longissimus dorsi.

Key words: sheep, alternative splicing, differentialsplicing gene, RNA-seq

鲍晶晶, 浦亚斌, 马月辉, 赵倩君. 绵羊不同发育阶段背最长肌组织中可变剪接的鉴定与分析[J]. 生物技术通报, 2019, 35(7): 33-38.

BAO Jing-jing, PU Ya-bin, MA Yue-hui, ZHAO Qian-jun. Identification and Analysis of Alternative Splicing in Longissimus dorsi of Sheep at Different Development Stages[J]. Biotechnology Bulletin, 2019, 35(7): 33-38.

参考文献

[1] Tress ML, Abascal F, Valencia A.Alternative splicing may not be the key to proteome complexity[J]. Trends Biochem Sci, 2017, 42(2):98-110.
[2] Baralle FE, Giudice J.Alternative splicing as a regulator of development and tissue identity[J]. Nat Rev Mol Cell Biol, 2017, 18(7):437-451.
[3] Blencowe BJ.The relationship between alternative splicing and proteomic complexity[J]. Trends Biochem Sci, 2017, 42(6):407-408.
[4] Nilsen TW, Graveley BR.Expansion of the eukaryotic proteome by alternative splicing[J]. Nature, 2010, 463(7280):457-463.
[5] Graveley BR.Alternative splicing:increasing diversity in the proteomic world[J]. Trends Genet, 2001, 17(2):100-107.
[6] Wang ET, Sandberg R, Luo S, et al.Alternative isoform regulation in human tissue transcriptomes[J]. Nature, 2008, 456(7221):470-476.
[7] Blue RE, Koushik A, Engels NM, et al.Modulation of alternative splicing of trafficking genes by genome editing reveals functional consequences in muscle biology[J]. Int J Biochem Cell Biol, 2018, 105:134-143.
[8] Kuranaga Y, Sugito N, Shinohara H, et al.SRSF3, a splicer of the PKM gene, regulates cell growth and maintenance of cancer-specific energy metabolism in colon cancer cells[J]. Int J Mol Sci, 2018, 19(10):3012.
[9] Pamela L, Antonella S, Stefania F, et al.RNA-binding proteins RBM20 and PTBP1 regulate the alternative splicing of FHOD3[J]. Int J Biochem Cell Biol, 2019, 106(1):74-83.
[10] Liu Y, Huang W, Gao X, et al.Regulation between two alternative splicing isoforms ZNF148(FL)and ZNF148(DeltaN), and their roles in the apoptosis and invasion of colorectal cancer[J]. Pathol Res Pract, 2019, 215(2):272-277.
[11] Nicholson P, Yepiskoposyan H, Metze S, et al.Nonsense-mediated mRNA decay in human cells:mechanistic insights, functions beyond quality control and the double-life of NMD factors[J]. Cell Mol Life Sci, 2010, 67(5):677-700.
[12] Yu S, Wang G, Liao J, et al.Five alternative splicing variants of the TYR gene and their different roles in melanogenesis in the Muchuan black-boned chicken[J]. Br Poult Sci, 2019, 60(1):8-14.
[13] Miao X, Luo Q, et al.Ovarian transcriptomic analysis reveals the alternative splicing events associated with fecundity in different sheep breeds[J]. Anim Reprod Sci, 2018, 198:177-183.
[14] 张天, 李祥龙, 周荣艳, 等. 不同毛色山羊皮肤组织Agouti基因剪接体类型研究[J]. 畜牧兽医学报, 2015(11):1934-1943.
[15] Shen S, Park JW, Lu ZX, et al.rMATS:robust and flexible detec-tion of differential alternative splicing from replicate RNA-Seq data[J]. Proc Natl Acad Sci USA, 2014, 111(51):E5593-E5601.
[16] Patel R K, Jain M.NGS QC Toolkit:a toolkit for quality control of next generation sequencing data[J]. PLoS One, 2012, 7(2):e30619.
[17] Kim D, Langmead B, Salzberg SL.HISAT:a fast spliced aligner with low memory requirements[J]. Nat Methods, 2015, 12(4):357-360.
[18] Pertea M, Pertea GM, Antonescu CM, et al.StringTie enables improved reconstruction of a transcriptome from RNA-seq reads[J]. Nat Biotechnol, 2015, 33(3):290-295.
[19] Trapnell C, Roberts A, Goff L, et al.Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks[J]. Nat Protoc, 2012, 7(3):562-578.
[20] Frazee AC, Pertea G, Jaffe AE, et al.Ballgown bridges the gap between transcriptome assembly and expression analysis[J]. Nat Biotechnol, 2015, 33(3):243-246.
[21] Pertea M, Kim D, Pertea GM, et al.Transcript-level expression analysis of RNA-seq experiments with HISAT, StringTie and Ballgown[J]. Nat Protoc, 2016, 11(9):1650-1667.
[22] Reimand J, Kull M, Peterson H, et al.g:Profiler--a web-based toolset for functional profiling of gene lists from large-scale experiments[J]. Nucleic Acids Res, 2007, 35:W193-200.
[23] Reimand J, Arak T, Vilo J. g:Profiler--a web server for functional interpretation of gene lists(2011 update)[J]. Nucleic Acids Res, 2011, 39:W307-315.
[24] Huang DW, Sherman BT, Lempicki RA.Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources[J]. Nat Protoc, 2009, 4(1):44-57.
[25] Huang DW, Sherman BT.Bioinformatics enrichment tools:paths toward the comprehensive functional analysis of large gene lists[J]. Nucleic Acids Res, 2009, 37(1):1-13.
[26] Gilbert W.Why genes in pieces?[J]. Nature, 1978, 271(5645):501.
[27] Climente-Gonzalez H, Porta-Pardo E, Godzik A, et al.The functional impact of alternative splicing in cancer[J]. Cell Rep, 2017, 20(9):2215-2226.
[28] Martinez-Montiel N, Rosas-Murrieta NH, Anaya RM, et al.Alternative splicing as a target for cancer treatment[J]. Int J Mol Sci, 2018, 19(2):545.
[29] Urbanski LM, Leclair N, Anczukow O.Alternative-splicing defects in cancer:splicing regulators and their downstream targets, guiding the way to novel cancer therapeutics[J]. Wiley Interdiscip Rev RNA, 2018, 9(4):e1476.
[30] Peterfy M, Phan J, Reue K.Alternatively spliced lipin isoforms exhibit distinct expression pattern, subcellular localization, and role in adipogenesis[J]. J Biol Chem, 2005, 280(38):32883-32889.
[31] Zhang X, Yan H, Wang K, et al.Goat CTNNB1:mRNA expression profile of alternative splicing in testis and association analysis with litter size[J]. Gene, 2018, 679:297-304.
[32] Zhang X, Zhou Y, Pan C, et al.Novel alternative splice variants of NFIX and their diverse mRNA expression patterns in dairy goat[J]. Gene, 2015, 569(2):250-258.
[33] 冉茂良, 陈斌, 李智, 等. 基于RNA-seq测序数据鉴定和分析猪基因组可变剪接事件[J]. 中国科学:生命科学, 2016(3):274-284.
[34] 张敏, 王杰, 孙艳发, 等. 肉鸡肌肉与脂肪组织基因组差异剪接基因分析[J]. 畜牧兽医学报, 2018(10):2124-2132.
[35] 郭家中, 陶海溪, 李鹏飞, 等. 简州大耳羊早期胎儿到新出生阶段背最长肌中可变剪接的鉴定与分析[J]. 西北农业学报, 2018(3):316-325.
[36] 黄艳群, 陈文, 李宁, 等. 鸡Lmbr1基因一种异常可变剪接的克隆和表达分析[J]. 畜牧兽医学报, 2010(5):518-523.
[37] Picard B, Lefaucheur L, et al.Muscle fibre ontogenesis in farm animal species[J]. Reprod Nutr Dev, 2002, 42(5):415-431.
[38] Li B, Dong C, Li P, et al.Identification of candidate genes associated with porcine meat color traits by genome-wide transcriptome analysis[J]. Sci Rep, 2016, 6:35224.
[39] Hooper JE.A survey of software for genome-wide discovery of differential splicing in RNA-Seq data[J]. Hum Genomics, 2014, 8:3. doi:10.1186/1479-7364-8-3.