杂交石斑鱼和母本褐点石斑鱼转录组测序及差异表达基因分析
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摘要
【目的】筛选褐点石斑鱼(Epinephelus fuscoguttatus)及其杂交子一代(E. fuscoguttatus♀×E. polyphekadion♂,F1)的差异表达基因,探讨褐点石斑鱼及F1代之间的生长差异性。【方法】采用Illumina HiSeq 2000测序平台对同龄的褐点石斑鱼及其杂交F1的脑、肝脏和肌肉组织进行转录组测序,通过edgeR软件包筛选差异表达基因,并对差异表达基因进行GO(Gene Ontology)功能注释和KOG注释,最后通过实时荧光定量PCR验证转录组数据。【结果】测序的原始数据经过质量控制和组装共得到54 610条unigenes。组装的unigenes共有28 832条unigenes得到注释,共获得20234条差异表达基因,其中有10218条上调,10016条下调。GO功能注释显示共34061条unigenes聚类到涉及生物过程、分子功能和细胞组分的53个功能类别,其中被较多基因聚类到的功能类别为细胞过程、结合、单一生物过程、代谢过程和催化活性等。KOG注释发现,42 753 unigenes被注释到25个功能类别中,其中较多涉及信号转导机制、基因功能预测、翻译后修饰,蛋白质周转和分子伴侣、转录等。进一步筛选到一批和生长相关的差异表达基因,如GH、GHRH、PRL、SLP、AVTp等,这些差异表达基因在F1中的表达水平均高于其亲本褐点石斑鱼,与杂交F1相较于褐点石斑鱼生长快速的性状相符。【结论】研究完成了对褐点石斑鱼及其杂交F1的转录组测序、组装,获得了测序数据在不同数据库的功能注释信息,同时筛选了一批生长相关差异表达基因,这些基因在F1中的表达水平均高于其亲本褐点石斑鱼,与现实生长性状相符。
【Objective】Significant difference in the growth of tiger grouper(Epinephelus fuscoguttatus)and its hybrid grouper(E. fuscoguttatus♀×E. polyphekadion♂)F1 was found in grouper aquaculture.To analyze the molecular mechanisms leading to the fast-growth of the hybrid grouper, this study aimed to characterize the differentially expressed genes of that have growth characters, which may provide a theoretical basis for the crossbreeding of grouper.【Methods】The Illumina Hi Seq 2000 sequencing platform was used for RNA sequencing of different tissues(the brain, liver and muscle)of tiger grouper and hybrid grouper F1 of the same age. Differentially expressed genes were screened and functional annotation were conducted. Finally, real-time quantitative-PCR was performed to verify the transcriptome data.【Results】The raw data of sequencing obtained a total of 54 610 unigenes after quality control and assembly. The unigenes were searched against Nr, Swissprot, KOG and KEGG databases by BlastX program(E value≤10-5), and a total of 28 832 unigenes were annotated. The edgeR software package was used for differential expression analysis among the sample. A total of 20234 unigenes were obtained, of which 10 218 were up-regulated and 10 016 were down-regulated. The Gene Ontology(GO)functional annotation showed that a total of 34 061 differentially expressed genes were clustered into 53 functional categories involving biological processes, molecular functions, and cellular components. The functional categories clustered most of the genes involved in cellular processes, binding, single-organism process, metabolic process, and catalytic activity, etc. Cluster of Orthologous Groups of proteins(KOG) analysis showed that 42 753 unigenes were annotated into 25 functional categories, most of which were involved in signal transduction mechanisms, general function prediction only, post-translational modifications, posttranslational modification, protein turnover,chaperones and transcription. The study further screened a number of growth-related genes, GH, GHRH,PRL, SLP and AVTp, etc. In this study, the expression levels of these differentially expressed genes in F1 were higher than those of their parents, which was consistent with the rapid growth of hybrid F1 compared to tiger grouper.【Conclusion】The transcriptome sequencing and assembly of tiger grouper and its hybrid F1 were completed. Functional annotation information of sequencing data in different databases was obtained. A batch of growth-related differentially expressed genes was screened, the expression level of these genes in F1 is higher than tiger grouper, which consistent with realistic growth traits.
【Objective】Significant difference in the growth of tiger grouper(Epinephelus fuscoguttatus)and its hybrid grouper(E. fuscoguttatus♀×E. polyphekadion♂)F1 was found in grouper aquaculture.To analyze the molecular mechanisms leading to the fast-growth of the hybrid grouper, this study aimed to characterize the differentially expressed genes of that have growth characters, which may provide a theoretical basis for the crossbreeding of grouper.【Methods】The Illumina Hi Seq 2000 sequencing platform was used for RNA sequencing of different tissues(the brain, liver and muscle)of tiger grouper and hybrid grouper F1 of the same age. Differentially expressed genes were screened and functional annotation were conducted. Finally, real-time quantitative-PCR was performed to verify the transcriptome data.【Results】The raw data of sequencing obtained a total of 54 610 unigenes after quality control and assembly. The unigenes were searched against Nr, Swissprot, KOG and KEGG databases by BlastX program(E value≤10-5), and a total of 28 832 unigenes were annotated. The edgeR software package was used for differential expression analysis among the sample. A total of 20234 unigenes were obtained, of which 10 218 were up-regulated and 10 016 were down-regulated. The Gene Ontology(GO)functional annotation showed that a total of 34 061 differentially expressed genes were clustered into 53 functional categories involving biological processes, molecular functions, and cellular components. The functional categories clustered most of the genes involved in cellular processes, binding, single-organism process, metabolic process, and catalytic activity, etc. Cluster of Orthologous Groups of proteins(KOG) analysis showed that 42 753 unigenes were annotated into 25 functional categories, most of which were involved in signal transduction mechanisms, general function prediction only, post-translational modifications, posttranslational modification, protein turnover,chaperones and transcription. The study further screened a number of growth-related genes, GH, GHRH,PRL, SLP and AVTp, etc. In this study, the expression levels of these differentially expressed genes in F1 were higher than those of their parents, which was consistent with the rapid growth of hybrid F1 compared to tiger grouper.【Conclusion】The transcriptome sequencing and assembly of tiger grouper and its hybrid F1 were completed. Functional annotation information of sequencing data in different databases was obtained. A batch of growth-related differentially expressed genes was screened, the expression level of these genes in F1 is higher than tiger grouper, which consistent with realistic growth traits.
引文
[1]ARAI T.Diversity and conservation of coral reef fishes in the malaysian south china sea[J].Reviews in Fish Biology&Fisheries,2015,25(1):85-101.
[2]罗鸣,陈傅晓,刘龙龙,等.我国石斑鱼养殖疾病的研究进展[J].水产科学,2013,32(9):549-554.
[3]JAMES C M,AL-THOBARTI S A,RASEM B M,et al.Potential of grouper hybrid(Epinephilus fuscoguttatus×E.polyphikadion)for aquaculture[J].ICLARM[International Center for Living Aquatic Resources Management]Quarterly,1999,22:19-23.
[4]SAROGLIA M,LIU Z.Functional genomics in aquaculture[M].Iowa:John Wiley&Sons,2012.
[5]SUN Y,GUO C Y,WANG D D,et al.Transcriptome analysis reveals the molecular mechanisms underlying growth superiority in a novel grouper hybrid(Epinephelus fuscogutatus♀×E.lanceolatus♂)[J].BMC Genetics,2016,17(1):24.
[6]WANG Y D,WANG Y H,HUI C F,et al.Transcriptome analysis of the effect of Vibrio alginolyticus infection on the innate immunity-related TLR5-mediated induction of cytokines in Epinephelus lanceolatus[J].Fish&Shellfish Immunology,2016,52:31-43.
[7]LIU C C,HO L P,YANG C H,et al.Comparison of grouper infection with two different iridoviruses using transcriptome sequencing and multiple reference species selection[J].Fish&Shellfish Immunology,2017,71:264-274.
[8]GAO Q,YUE Y,MIN M,et al.Time-series transcriptomic analysis of the kelp grouper Epinephelus moara in response to low salinity stress[J].Animal cells and systems,2018,22(4):234-242.
[9]ANDERSON K,KUO C Y,LU M W,et al.Atranscriptomic investigation of digestive processes in orange-spotted grouper,Epinephelus coioides,before,during,and after metamorphic development[J].Gene,2018,661:95-108.
[10]QIU F,QU M,ZHANG X,et al.Hypothalamus and pituitary transcriptome profiling of male and female Hong Kong grouper(Epinephelus akaara)[J].Gene,2018,656:73-79.
[11]GRABHERR M G,HAAS B J,YASSOUR M,et al.Full-length transcriptome assembly from RNA-Seq data without a reference genome[J].Nature Biotechnology,2011,29(7):644-652.
[12]HAAS B J,PAPANICOLAOU A,YASSOUR M,et al.De novo transcript sequence reconstruction from RNA-Seq:reference generation and analysis with trinity[J].Nature Protocols,2013,8(8):1494-1512.
[13]CONESA A,G?TZ S,GARCíA GóMEZ J M,et al.Blast2GO:a universal tool for annotation,visualization and analysis in functional genomics research[J].Bioinformatics,2005,21(18):3674-3676.
[14]ISELI C,JONGENEEL C V,BUCHER P.ESTS can:Aprogram for detecting,evaluating,and reconstructing potential coding regions in EST sequences[J].Proceedings/.International Conference on Intelligent Systems for Molecular Biology;ISMB.International Conference on Intelligent Systems for Molecular Biology,1999,99:138-148.
[15]MORTAZAVI A,WILLIAMS B A,MCCUE K,et al.Mapping and quantifying mammalian transcriptomes by RNA-Seq[J].Nature Methods,2008,5(7):621-628.
[16]Xie C,Mao X,Huang J,et al.KOBAS 2.0:a web server for annotation and identification of enriched pathways and diseases[J].Nucleic acids research,2011,39(suppl_2):W316-W322.
[17]CAI Y,WANG S,GUO W,et al.Transcriptome analysis provides insights into the immune responsive pathways and genes in the head kidney of tiger grouper(Epinephelus fuscoguttatus)fed with Spatholobus suberectus,Phellodendron amurense,or Eclipta prostrata[J].Fish&Shellfish Immunology,2018,73:100-111.
[18]LU M W,NGOU F H,CHAO Y M,et al.Transcriptome characterization and gene expression of Epinephelus spp in endoplasmic reticulum stress-related pathway during betanodavirus infection in vitro[J].Bmc Genomics,2012,13(1):651.
[19]HOU R,BAO Z,WANG S,et al.Transcriptome sequencing and de novo analysis for yesso scallop(Patinopecten yessoensis)using 454 GS FLX[J].Plos One,2011,6(6):e21560.
[20]汝少国,郑艳.外源化学物质对鱼类生长和GH/IGF-I轴的影响[J].中国海洋大学学报(自然科学版),2012(S1):102-106.
[21]NAM B H,MOON J Y,KIM Y O,et al.Molecular and functional analyses of growth hormone-releasing hormone(GHRH)from olive flounder(Paralichthys olivaceus)[J].Comparative Biochemistry&Physiology Part B Biochemistry&Molecular Biology,2011,159(2):84-91.
[22]刘金亮,梁利群.鱼类催乳素及其在渗透压调节中的作用[J].水产学杂志,2011,24(1):50-54.
[23]RAND-WEAVER M,NOSO T,MURAMOTO K,et al.Isolation and characterization of somatolactin,a new protein related to growth hormone and prolactin from Atlantic cod(Gadus morhua)pituitary glands[J].Biochemistry,1991,30(6):1509-1515.
[24]LEE S,LIM B,LEE J,et al.Up-regulation of the arginine vasotocin precursor gene from Paralichthys olivaceus:isolation and expression upon acute pathogen invasion[J].Genes&Genomics,2014,36(4):443-453.
[25]LEE S K,MOON J W,LEE Y W,et al.The effect of high glucose levels on the hypermethylation of protein phosphatase 1 regulatory subunit 3C(PPP1R3C)gene in colorectal cancer[J].Journal of Genetics,2015,94(1):75-85.
[26]HER G M,CHIANG C C,CHEN W Y,et al.In vivo studies of liver-type fatty acid binding protein(L-FABP)gene expression in liver of transgenic zebrafish(Danio rerio)[J].Febs Letters,2003,538(1/2/3):125-133.
[27]JIANG S,HELLER B,TAGLIABRACCI V S,et al.Starch binding domain-containing protein 1/genethonin1 is a novel participant in glycogen metabolism[J].Journal of Biological Chemistry,2010,285(46):34960-34971.
[28]SUGIYAMA M,TAKENAGA F,KITANI Y,et al.Homozygous and heterozygous GH transgenesis alters fatty acid composition and content in the liver of Amago salmon(Oncorhynchus masou ishikawae)[J].Biology Open,2012,1(10):1035-1042.
[29]FARIN H F,MANSOURI A,PETRY M,et al.T-box protein tbx18 interacts with the paired box protein pax3in the development of the paraxial mesoderm[j].journal of biological chemistry,2008,283(37):25372-25380.
[30]JAYNES J B,JOHNSON J E,BUSKIN J N,et al.The muscle creatine kinase gene is regulated by multiple upstream elements,including a muscle-specific enhancer[J].Molecular and Cellular Biology,1988,8(1):62-70.
[31]PARMACEK M S.Myocardin-Related transcription factors:critical coactivators regulating cardiovascular development and adaptation[J].Circulation Research,2007,100(5):633-644.
[32]RUDNICKI M A,SCHNEGELSBERG P N J,STEAD RH,et al.MyoD or Myf-5 is required for the formation of skeletal muscle[J].Cell,1993,75(7):1351-1359.
[2]罗鸣,陈傅晓,刘龙龙,等.我国石斑鱼养殖疾病的研究进展[J].水产科学,2013,32(9):549-554.
[3]JAMES C M,AL-THOBARTI S A,RASEM B M,et al.Potential of grouper hybrid(Epinephilus fuscoguttatus×E.polyphikadion)for aquaculture[J].ICLARM[International Center for Living Aquatic Resources Management]Quarterly,1999,22:19-23.
[4]SAROGLIA M,LIU Z.Functional genomics in aquaculture[M].Iowa:John Wiley&Sons,2012.
[5]SUN Y,GUO C Y,WANG D D,et al.Transcriptome analysis reveals the molecular mechanisms underlying growth superiority in a novel grouper hybrid(Epinephelus fuscogutatus♀×E.lanceolatus♂)[J].BMC Genetics,2016,17(1):24.
[6]WANG Y D,WANG Y H,HUI C F,et al.Transcriptome analysis of the effect of Vibrio alginolyticus infection on the innate immunity-related TLR5-mediated induction of cytokines in Epinephelus lanceolatus[J].Fish&Shellfish Immunology,2016,52:31-43.
[7]LIU C C,HO L P,YANG C H,et al.Comparison of grouper infection with two different iridoviruses using transcriptome sequencing and multiple reference species selection[J].Fish&Shellfish Immunology,2017,71:264-274.
[8]GAO Q,YUE Y,MIN M,et al.Time-series transcriptomic analysis of the kelp grouper Epinephelus moara in response to low salinity stress[J].Animal cells and systems,2018,22(4):234-242.
[9]ANDERSON K,KUO C Y,LU M W,et al.Atranscriptomic investigation of digestive processes in orange-spotted grouper,Epinephelus coioides,before,during,and after metamorphic development[J].Gene,2018,661:95-108.
[10]QIU F,QU M,ZHANG X,et al.Hypothalamus and pituitary transcriptome profiling of male and female Hong Kong grouper(Epinephelus akaara)[J].Gene,2018,656:73-79.
[11]GRABHERR M G,HAAS B J,YASSOUR M,et al.Full-length transcriptome assembly from RNA-Seq data without a reference genome[J].Nature Biotechnology,2011,29(7):644-652.
[12]HAAS B J,PAPANICOLAOU A,YASSOUR M,et al.De novo transcript sequence reconstruction from RNA-Seq:reference generation and analysis with trinity[J].Nature Protocols,2013,8(8):1494-1512.
[13]CONESA A,G?TZ S,GARCíA GóMEZ J M,et al.Blast2GO:a universal tool for annotation,visualization and analysis in functional genomics research[J].Bioinformatics,2005,21(18):3674-3676.
[14]ISELI C,JONGENEEL C V,BUCHER P.ESTS can:Aprogram for detecting,evaluating,and reconstructing potential coding regions in EST sequences[J].Proceedings/.International Conference on Intelligent Systems for Molecular Biology;ISMB.International Conference on Intelligent Systems for Molecular Biology,1999,99:138-148.
[15]MORTAZAVI A,WILLIAMS B A,MCCUE K,et al.Mapping and quantifying mammalian transcriptomes by RNA-Seq[J].Nature Methods,2008,5(7):621-628.
[16]Xie C,Mao X,Huang J,et al.KOBAS 2.0:a web server for annotation and identification of enriched pathways and diseases[J].Nucleic acids research,2011,39(suppl_2):W316-W322.
[17]CAI Y,WANG S,GUO W,et al.Transcriptome analysis provides insights into the immune responsive pathways and genes in the head kidney of tiger grouper(Epinephelus fuscoguttatus)fed with Spatholobus suberectus,Phellodendron amurense,or Eclipta prostrata[J].Fish&Shellfish Immunology,2018,73:100-111.
[18]LU M W,NGOU F H,CHAO Y M,et al.Transcriptome characterization and gene expression of Epinephelus spp in endoplasmic reticulum stress-related pathway during betanodavirus infection in vitro[J].Bmc Genomics,2012,13(1):651.
[19]HOU R,BAO Z,WANG S,et al.Transcriptome sequencing and de novo analysis for yesso scallop(Patinopecten yessoensis)using 454 GS FLX[J].Plos One,2011,6(6):e21560.
[20]汝少国,郑艳.外源化学物质对鱼类生长和GH/IGF-I轴的影响[J].中国海洋大学学报(自然科学版),2012(S1):102-106.
[21]NAM B H,MOON J Y,KIM Y O,et al.Molecular and functional analyses of growth hormone-releasing hormone(GHRH)from olive flounder(Paralichthys olivaceus)[J].Comparative Biochemistry&Physiology Part B Biochemistry&Molecular Biology,2011,159(2):84-91.
[22]刘金亮,梁利群.鱼类催乳素及其在渗透压调节中的作用[J].水产学杂志,2011,24(1):50-54.
[23]RAND-WEAVER M,NOSO T,MURAMOTO K,et al.Isolation and characterization of somatolactin,a new protein related to growth hormone and prolactin from Atlantic cod(Gadus morhua)pituitary glands[J].Biochemistry,1991,30(6):1509-1515.
[24]LEE S,LIM B,LEE J,et al.Up-regulation of the arginine vasotocin precursor gene from Paralichthys olivaceus:isolation and expression upon acute pathogen invasion[J].Genes&Genomics,2014,36(4):443-453.
[25]LEE S K,MOON J W,LEE Y W,et al.The effect of high glucose levels on the hypermethylation of protein phosphatase 1 regulatory subunit 3C(PPP1R3C)gene in colorectal cancer[J].Journal of Genetics,2015,94(1):75-85.
[26]HER G M,CHIANG C C,CHEN W Y,et al.In vivo studies of liver-type fatty acid binding protein(L-FABP)gene expression in liver of transgenic zebrafish(Danio rerio)[J].Febs Letters,2003,538(1/2/3):125-133.
[27]JIANG S,HELLER B,TAGLIABRACCI V S,et al.Starch binding domain-containing protein 1/genethonin1 is a novel participant in glycogen metabolism[J].Journal of Biological Chemistry,2010,285(46):34960-34971.
[28]SUGIYAMA M,TAKENAGA F,KITANI Y,et al.Homozygous and heterozygous GH transgenesis alters fatty acid composition and content in the liver of Amago salmon(Oncorhynchus masou ishikawae)[J].Biology Open,2012,1(10):1035-1042.
[29]FARIN H F,MANSOURI A,PETRY M,et al.T-box protein tbx18 interacts with the paired box protein pax3in the development of the paraxial mesoderm[j].journal of biological chemistry,2008,283(37):25372-25380.
[30]JAYNES J B,JOHNSON J E,BUSKIN J N,et al.The muscle creatine kinase gene is regulated by multiple upstream elements,including a muscle-specific enhancer[J].Molecular and Cellular Biology,1988,8(1):62-70.
[31]PARMACEK M S.Myocardin-Related transcription factors:critical coactivators regulating cardiovascular development and adaptation[J].Circulation Research,2007,100(5):633-644.
[32]RUDNICKI M A,SCHNEGELSBERG P N J,STEAD RH,et al.MyoD or Myf-5 is required for the formation of skeletal muscle[J].Cell,1993,75(7):1351-1359.