虹鳟肝组织新转录本分析及基因结构优化
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摘要
目的虹鳟热应激下肝RNA-seq数据中新转录本的分析及已注释基因结构优化。方法以虹鳟肝为材料提取总RNA,构建cDNA文库,并利用Illumina双端测序Hiseq 2500平台进行测序。运用Cufflinks软件对测序数据进行组装,将其与虹鳟参考基因组进行序列比对。结果发掘新转录本6555个,其中30个新转录本在热应激前后差异表达(P<0.05)。与GO数据库比对对新转录本进行功能注释,获得3097个新转录本的注释。与KEGG数据库比对,共有3617个新转录本注释到284条代谢通路中。对19 424个已注释基因的结构进行优化,延伸了14 719个基因的5′端和14 796个基因的3′端。结论通过对发掘的6555个新转录本分析,并对19 424个已注释基因结构优化,为虹鳟基因组注释信息的完善提供了有力的借鉴,并为进一步了解虹鳟热应激的机制提供更有力的理论基础。
Objective To discover novel transcripts from RNA-seq data of rainbow trout under heat stress and optimize the annotated gene structure. Methods Total RNA was extracted from the liver of rainbow trout and used to construct a cDNA library that was sequenced by the Illumina two-terminal sequencing Hiseq 2500 platform. Sequencing data were assembled by Cufflinks software and aligned with the rainbow trout genome. Results Among 6555 new transcripts, 30 were differentially expressed under heat stress(P < 0.05). A total of 3097 novel transcripts were annotated and blasted by the GO database. There were 3617 new transcripts annotated in 284 metabolic pathways by KEGG database. The structure of 19 424 annotated genes was optimized, and the 5′-ends of 14 719 genes and 3′-ends of 14 796 genes were extended. Conclusions Discovery and analysis of 6555 novel transcripts and 19 424 gene structure optimization provide a powerful reference optimizing the genome annotation information of rainbow trout, and a more powerful theoretical basis for further understanding the mechanism of heat stress in rainbow trout.
Objective To discover novel transcripts from RNA-seq data of rainbow trout under heat stress and optimize the annotated gene structure. Methods Total RNA was extracted from the liver of rainbow trout and used to construct a cDNA library that was sequenced by the Illumina two-terminal sequencing Hiseq 2500 platform. Sequencing data were assembled by Cufflinks software and aligned with the rainbow trout genome. Results Among 6555 new transcripts, 30 were differentially expressed under heat stress(P < 0.05). A total of 3097 novel transcripts were annotated and blasted by the GO database. There were 3617 new transcripts annotated in 284 metabolic pathways by KEGG database. The structure of 19 424 annotated genes was optimized, and the 5′-ends of 14 719 genes and 3′-ends of 14 796 genes were extended. Conclusions Discovery and analysis of 6555 novel transcripts and 19 424 gene structure optimization provide a powerful reference optimizing the genome annotation information of rainbow trout, and a more powerful theoretical basis for further understanding the mechanism of heat stress in rainbow trout.
引文
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[2] Chen C,Ai H,Ren J,et al.A global view of porcine transcriptome in three tissues from a full-sib pair with extreme phenotypes in growth and fat deposition by paired-end RNA sequencing [J].BMC Genomics,2011,12:448.
[3] J?ger M,Ott CE,Grünhagen J,et al.Composite transcriptome assembly of RNA-Seq data in a sheep model for delayed bone healing [J].BMC Genomics,2011,12:158.
[4] 夏斌鹏,刘哲,周彦静,等.慢性热应激对虹鳟部分血清非特异性免疫指标的影响[J]农业生物技术学报,2017,25(7):1078-1085.Xia BP,Liu Z,Zhou YZ,et al.Effects of chronic heat stress on part of serum non-specific immunity parameters in rainbow trout (Oncorhynchus mykiss) [J].J Agric Biotechnol,25(7):1078-1085.
[5] Lewis JW,Hori TS,Rise ML,et al.Transcriptome responses to heat stress in the nucleated red blood cells of the rainbow trout (Oncorhynchus mykiss) [J].Physiol Genomics,2010,42(3):361-373.
[6] Verleih M,Borchel A,Krasnov A,et al.Impact of thermal stress on kidney-specific gene expression in farmed regional and imported rainbow trout [J].Mar Biotechnol,2015,17(5):576-592.
[7] Li YJ,Huang JQ,Liu Z.et al.Transcriptome analysis provides insights into hepatic responses to moderate heat stress in the rainbow trout (Oncorhynchus mykiss) [J].Gene,2017,1119(17):30231-30237.
[8] Wang Y,Xu J,Sheng L.Field and laboratory investigations of the thermal influence on tissue-specific Hsp70 levels in common carp (Cyprinus carpio) [J].Comp Biochem Physiol A Mol Integr Physiol,2007,148(4):821-827.
[9] Fangue NA,Hofmeister M,Schulte PM.Intraspecific variation in thermal tolerance and heat shock protein gene expression in common killifish,Fundulus heteroclitus [J].J Exp Biol,2006,209(15):2859-2872.
[10] Molina A,Biemar F,Müller F,et al.Cloning and expression analysis of an inducible HSP70 gene from tilapia fish [J].FEBS Lett,2000,474(1):5-10.
[11] Li Z,Liu Z,Wang YN,et al.Effects of heat stress on serum cortisol,alkaline phosphatase activity and heat shock protein 40 and 90βmRNA expression in rainbow trout Oncorhynchus mykiss [J].Biologia,2016,71(1):109-115.
[12] Wang Y,Liu Z,Li Z,et al.Effects of heat stress on respiratory burst,oxidative damage and SERPINH1 (HSP47) mRNA expression in rainbow trout Oncorhynchus mykiss [J].Fish Physiol Biochem,2016,42(2):701-710.
[13] Shi HN,Liu Z,Zhang JP,et al.Effect of heat stress on heat-shock protein (Hsp60) mRNA expression in rainbow trout Oncorhynchus mykiss [J].Genet Mol Res,2015,14(2):5280- 5286.
[14] Boone AN,Vijayan MM.Constitutive heat shock protein 70 (HSC70) expression in rainbow trout hepatocytes:effect of heat shock and heavy metal exposure [J].Comp Biochem Physiol C Toxicol Pharmacol,2002,132(2):223-233.
[15] Heredia-Middleton P,Brunelli J,Drew RE,et al.Heat shock protein (HSP70) RNA expression differs among rainbow trout (Oncorhynchus mykiss) clonal lines [J].Comp Biochem Physiol B Biochem Mol Biol,2008,149(4):552-556.
[16] 秦彩艳,霍金龙,王配,等.猪新基因KCNJ12编码区电子克隆、RT-PCR验证和组织表达分析[J].华北农学报,2014,29(1):41-49.Qin CY,Huo JL,Wang P,et al.In silico cloning,RT-PCR verification and tissues expression of coding region sequence of novel swine gene KCNJ12 [J].Acta Agric Boreali Sin,2014,29(1):41-49.
[17] Currie S,LeBlanc S,Watters MA,et al.Agonistic encounters and cellular angst:social interactions induce heat shock proteins in juvenile salmonid fish [J].Proc Biol Sci,2010,277(1683):905-913.
[18] Ojima N.Rainbow trout hspb1 (hsp27):identification of two mRNA splice variants that show predominant expression in muscle tissues [J].Comp Biochem Physiol B Biochem Mol Biol,2007,148(3):277-285.