角鳖RNA-seq转录组分析及生长相关基因筛选
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摘要
角鳖是一种具有极高经济价值的淡水动物,市场开发潜力较大。其生长上存在的显著差异,制约了角鳖养殖业的健康可持续发展。为了探索角鳖生长发育的相关分子机制,本研究以生长存在显著差异的快长组和慢长组角鳖肝脏为材料,采用Illumina HiSeq TM 2500高通量测序平台进行转录组分析,经过拼接组装共获得264 757 372条Clean reads,148 093个Unigene,序列平均长度为900.8 bp。将Unigene与NR、SWISSPROT、KOG、KEGG和GO进行比对,共有34 666条得到了注释结果。通过以快长组为对照组,慢长组为实验组进行基因表达情况对比,结果显示有2 246个基因表达存在差异,其中显著性上调的基因1 264个,显著性下调的基因982个。通过KEGG pathway分析,Fox O信号通路、p53信号通路、PI3K-Akt信号通路、细胞凋亡、TGF-beta信号通路及胰岛素信号通路等生长发育相关代谢途径的部分基因表达存在显著的差异。本研究为进一步揭示角鳖生长发育的相关分子机制、生长相关基因克隆和表达等研究及奠定了坚实的基础,也为后续开展龟鳖类分子生物学研究提供了宝贵的基因数据来源。
Apalone spinifera is a kind of freshwater animal with high economic value,and has great potential for market development. The great differences in their growth restrict the healthy and sustainable development of Apalone spinifera breeding industry. In order to explore the molecular mechanism related to the growth in Apalone spinifera, we took the liver of Apalone spinifera in fast growth group and slow growth group with great differences as materials to analyze their transcriptions by using Illumina HiSeqTM2500 high-throughput sequencing platform.In total 264 757 372 clean reads and 148 093 Unigenes were obtained through splicing and assembling, with an average sequence length of 900.8 bp. Comparing Unigene with NR, SWISSPROT, KOG, KEGG and GO, a total of 34 666 annotated results were obtained. Comparing slow growth group with fast growth group in gene expression,results showed that there were 2 246 differentially expressed genes, of which 1 264 were significantly up-regulated and 982 were significantly down-regulated. KEGG pathway analysis showed that there were significant differences in expression of some genes of the growth and development-related metabolic pathway like Foxo signaling pathway, p53 signaling pathway, PI3K-Akt signaling pathway, cell apoptosis, TGF-beta signaling pathway and insulin signaling pathway. This study could lay a solid foundation for further revealing the molecular mechanism of growth and development, cloning and expression of growth related genes in Apalone spinifera, and it would provide a valuable source of genetic data in molecular biology research for turtles.
Apalone spinifera is a kind of freshwater animal with high economic value,and has great potential for market development. The great differences in their growth restrict the healthy and sustainable development of Apalone spinifera breeding industry. In order to explore the molecular mechanism related to the growth in Apalone spinifera, we took the liver of Apalone spinifera in fast growth group and slow growth group with great differences as materials to analyze their transcriptions by using Illumina HiSeqTM2500 high-throughput sequencing platform.In total 264 757 372 clean reads and 148 093 Unigenes were obtained through splicing and assembling, with an average sequence length of 900.8 bp. Comparing Unigene with NR, SWISSPROT, KOG, KEGG and GO, a total of 34 666 annotated results were obtained. Comparing slow growth group with fast growth group in gene expression,results showed that there were 2 246 differentially expressed genes, of which 1 264 were significantly up-regulated and 982 were significantly down-regulated. KEGG pathway analysis showed that there were significant differences in expression of some genes of the growth and development-related metabolic pathway like Foxo signaling pathway, p53 signaling pathway, PI3K-Akt signaling pathway, cell apoptosis, TGF-beta signaling pathway and insulin signaling pathway. This study could lay a solid foundation for further revealing the molecular mechanism of growth and development, cloning and expression of growth related genes in Apalone spinifera, and it would provide a valuable source of genetic data in molecular biology research for turtles.
引文
Cai Y.F.,and Jia Z.J.,2013,Progress in environmental transcriptomics based on next-generation high-throughput sequencing,Shengwu Duoyangxing(Biodiversity Science),21(4):401-410(蔡元锋,贾仲君,2013,基于新一代高通量测序的环境微生物转录组学研究进展,生物多样性,21(4):401-410)
Grabherr M.M.M.G.,Haas B.B.B.J.,Yassour M.,Levin J.J.J.Z.,and Thompson D.D.D.A.,2011,Trinity:reconstructing a full-length transcriptome without a genome from RNA-Seq data,Nature Biotechnology,29(7):644-652
Jiang F.X.,Jiang X.Y.,Wu S.,Yang L.J.,Li W.Y.,and Chen Q.B.,2015,Transcriptome sequencing and characteristic analysis of Ornithogalum caudatum,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),13(9):2038-2050(姜福星,江欣燕,吴生,杨丽娟,李文骥,陈其兵,2015,虎眼万年青鳞茎转录组特性分析,分子植物育种,13(9):2038-2050)
Langmead B.,and Salzburg S.S.S.L.,2012,Fast gapped-read alignment with Bowtie 2,Nature Methods,9(4):357
Liu H.L.,Zheng L.M.,Liu Q.Q.,Quan F.S.,and Zhang Y.,2013,Studies on the transcriptomes of non-model organisms,Yichuan(Hereditas),35(8):955-970(刘红亮,郑丽明,刘青青,权富生,张涌,2013,非模式生物转录组研究,遗传,35(8):955-970)
Luo H.,Ye H.,Xiao S.J.,Zheng S.M.,Wang X.Q.,and Wang Z.Y.,2015,Application of transcriptomics technology to aquatic animal research,Shuichan Xuebao(Journal of Fisheries of China),39(4):598-607(罗辉,叶华,肖世俊,郑曙明,王晓清,王志勇,2015,转录组学技术在水产动物研究中的运用,水产学报,39(4):598-607)
Pan Z.,Liu X.C.,and Zhang M.C.,2010.,Biological characteristics and artificial breeding technology of Apalone spinifera,Zhongguo Shuichan(China Fisheries),(7):50-51(潘志,刘旭成,张孟才,2010,角鳖的生物学特性及人工养殖技术,中国水产,(7):50-51)
Pertea G.,Huang X.,Liang F.,Antonescu V.,Sultana R.,Karamycheva S.,Lee Y.,White J.,Cheung F.,and Parvizi B.,2003,TIGR Gene Indices clustering tools(TGICL):a soft ware system for fast clustering of large EST datasets,Bioinformatics,19(5):651-652
Plummer M.V.,and Mills N.N.E.,2015,Growth and maturity of spiny softshell turtles(Apalone spinifera)in a small urban stream,Herpetological Conservation&Biology,10(2):688-694
Roberts A.,and Pachter L.,2013,Streaming fragment assignment for real-time analysis of sequencing experiments,Nature Methods,10(1):71-99
Wang J.,Wang Q.,Huang J.X.,and Qi R.L.,2016,Expression patterns and regulation mechanisms of microRNAs relate in growth and development of muscle in animals,Dongwu Yingyang Xuebao(Chinese Journal of Animal Nutrition),28(3):687-694(王敬,王琪,黄金秀,齐仁立,2016,动物肌肉生长发育相关microRNAs的表达模式和调控机制,动物营养学报,28(3):687-694)
Yao N.,Liu X.M.,Dong Y.Y.,Wang N.,Meng L.L.,and Li H.Y.,2017,Advances in application and sequencing methods of transcriptome,Beifang Yuanyi(Northern Horticulture),(12):192-198(姚娜,刘秀明,董园园,王南,孟璐璐,李海燕,2017,转录组的测序方法及应用研究概述,北方园艺,(12):192-198)
Yin Y.P.,Zhang Y.Q.,Shen Y.H.,Zhao Z.Y.,Xie S.Q.,Yang S.C.,and Liang Q.,2015,Screening of soybean genotype with high phosphorus efficiency using root morphology and architecture,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),13(5):999-1008(尹元萍,张雅琼,申毓晗,赵志勇,谢世清,杨生超,梁泉,2015,利用根系形态构型筛选磷高效大豆基因型,分子植物育种,13(5):999-1008)
Yue G.D.,Gao Q.,Luo L.H.,Wang J.Y.,Xu J.H.,and Yin Y.,2012,The application of high-throughput sequencing technology in plant and animal research,Zhongguo Kexue Shengming Kexue(Scientia Sinica(Vitae)),42(2):107-124(岳桂东,高强,罗龙海,王军一,许姣卉,尹烨,2012,高通量测序技术在动植物研究领域中的应用,中国科学:生命科学,42(2):107-124)
Grabherr M.M.M.G.,Haas B.B.B.J.,Yassour M.,Levin J.J.J.Z.,and Thompson D.D.D.A.,2011,Trinity:reconstructing a full-length transcriptome without a genome from RNA-Seq data,Nature Biotechnology,29(7):644-652
Jiang F.X.,Jiang X.Y.,Wu S.,Yang L.J.,Li W.Y.,and Chen Q.B.,2015,Transcriptome sequencing and characteristic analysis of Ornithogalum caudatum,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),13(9):2038-2050(姜福星,江欣燕,吴生,杨丽娟,李文骥,陈其兵,2015,虎眼万年青鳞茎转录组特性分析,分子植物育种,13(9):2038-2050)
Langmead B.,and Salzburg S.S.S.L.,2012,Fast gapped-read alignment with Bowtie 2,Nature Methods,9(4):357
Liu H.L.,Zheng L.M.,Liu Q.Q.,Quan F.S.,and Zhang Y.,2013,Studies on the transcriptomes of non-model organisms,Yichuan(Hereditas),35(8):955-970(刘红亮,郑丽明,刘青青,权富生,张涌,2013,非模式生物转录组研究,遗传,35(8):955-970)
Luo H.,Ye H.,Xiao S.J.,Zheng S.M.,Wang X.Q.,and Wang Z.Y.,2015,Application of transcriptomics technology to aquatic animal research,Shuichan Xuebao(Journal of Fisheries of China),39(4):598-607(罗辉,叶华,肖世俊,郑曙明,王晓清,王志勇,2015,转录组学技术在水产动物研究中的运用,水产学报,39(4):598-607)
Pan Z.,Liu X.C.,and Zhang M.C.,2010.,Biological characteristics and artificial breeding technology of Apalone spinifera,Zhongguo Shuichan(China Fisheries),(7):50-51(潘志,刘旭成,张孟才,2010,角鳖的生物学特性及人工养殖技术,中国水产,(7):50-51)
Pertea G.,Huang X.,Liang F.,Antonescu V.,Sultana R.,Karamycheva S.,Lee Y.,White J.,Cheung F.,and Parvizi B.,2003,TIGR Gene Indices clustering tools(TGICL):a soft ware system for fast clustering of large EST datasets,Bioinformatics,19(5):651-652
Plummer M.V.,and Mills N.N.E.,2015,Growth and maturity of spiny softshell turtles(Apalone spinifera)in a small urban stream,Herpetological Conservation&Biology,10(2):688-694
Roberts A.,and Pachter L.,2013,Streaming fragment assignment for real-time analysis of sequencing experiments,Nature Methods,10(1):71-99
Wang J.,Wang Q.,Huang J.X.,and Qi R.L.,2016,Expression patterns and regulation mechanisms of microRNAs relate in growth and development of muscle in animals,Dongwu Yingyang Xuebao(Chinese Journal of Animal Nutrition),28(3):687-694(王敬,王琪,黄金秀,齐仁立,2016,动物肌肉生长发育相关microRNAs的表达模式和调控机制,动物营养学报,28(3):687-694)
Yao N.,Liu X.M.,Dong Y.Y.,Wang N.,Meng L.L.,and Li H.Y.,2017,Advances in application and sequencing methods of transcriptome,Beifang Yuanyi(Northern Horticulture),(12):192-198(姚娜,刘秀明,董园园,王南,孟璐璐,李海燕,2017,转录组的测序方法及应用研究概述,北方园艺,(12):192-198)
Yin Y.P.,Zhang Y.Q.,Shen Y.H.,Zhao Z.Y.,Xie S.Q.,Yang S.C.,and Liang Q.,2015,Screening of soybean genotype with high phosphorus efficiency using root morphology and architecture,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),13(5):999-1008(尹元萍,张雅琼,申毓晗,赵志勇,谢世清,杨生超,梁泉,2015,利用根系形态构型筛选磷高效大豆基因型,分子植物育种,13(5):999-1008)
Yue G.D.,Gao Q.,Luo L.H.,Wang J.Y.,Xu J.H.,and Yin Y.,2012,The application of high-throughput sequencing technology in plant and animal research,Zhongguo Kexue Shengming Kexue(Scientia Sinica(Vitae)),42(2):107-124(岳桂东,高强,罗龙海,王军一,许姣卉,尹烨,2012,高通量测序技术在动植物研究领域中的应用,中国科学:生命科学,42(2):107-124)