Trend analysis of differentially expressed genes in gill of Exopalaemon carinicauda under hypoxia-reoxygenation stress
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摘要: 【目的】探究脊尾白虾（Exopalaemon carinicauda）鳃组织在渐变式低氧—复氧胁迫下的分子调控机制，为今后开展脊尾白虾耐低氧品系（种）的选育提供理论指导。【方法】通过模拟自然低氧环境的形成过程，分别于低氧处理0（对照）、3和6 h及复氧后1和8 h采集脊尾白虾鳃组织，利用Illumina HiSeqTM 4000测序平台进行转录组测序分析，经过滤和Trinity组装获得Unigenes，选取Nr、Swiss-Prot、KEGG和COG/KOG等数据库进行注释分析，在Omicsmart平台上完成差异表达基因筛选及其表达趋势分析，然后进行GO功能注释分析和KEGG信号通路富集分析，并随机选取5个差异表达基因进行实时荧光定量PCR验证。【结果】脊尾白虾鳃组织样本转录组测序数据经过滤后的Cleanreads进行Trinity组装共获得93227条Unigenes，其长度范围在201~35402 bp，平均长度为834 bp，N50长度为1352 bp。通过组间两两比较分析，共鉴定出4750个差异表达基因，其中上调差异表达基因3557个、下调差异表达基因2829个；超过50%的差异表达基因被显著富集到6种基因表达趋势模式中（P<0.01），具体表现为：Profile 0模式富集到415个基因，Profile 5模式富集到201个基因，Profile 11模式富集到371个基因，Profile 13模式富集到841个基因，Profile 17模式富集到387个基因，Profile 18模式富集到411个基因。6种基因表达趋势模式中的差异表达基因被注释到代谢进程、细胞进程、单一有机体进程、细胞、细胞零件、大分子复合物及催化活性等GO功能条目上；而KEGG信号通路富集分析结果显示，以Profile 13模式中的差异表达基因富集到最多信号通路（86条），其中呈显著富集的有8条，分别为核糖体、碳代谢、氧化磷酸化、氨基酸生物合成、内质网蛋白质加工、糖酵解/糖异生、谷胱甘肽代谢和蛋白输出。【结论】脊尾白虾鳃组织在受低氧胁迫早期通过合成蛋白质及提高代谢能力来抵御低氧环境，随着低氧胁迫时间的延长，物质合成和能量代谢活动均显著下降；但在复氧后随着复氧时间的延长，其蛋白质合成和能量代谢水平又逐渐升高恢复至常氧水平。Abstract: 【Objective】To investigate the molecular regulation mechanisms in gill tissue of Exopalaemon carinicauda under gradually changing hypoxia-reoxygenation stress,so as to provide theoretical reference for the breeding of hypoxia tolerance strains(species)of E. carinicauda.【Method】By simulating the formation process of hypoxia in the natural environment,gill tissues were collected at 0(control),3 and 6 h,and 1 and 8 h after reoxygenation,respectively. Transcriptome sequencing analysis was performed using Illumina HiSeqTM 4000 sequencing platform. Unigenes were obtained by filtration and Trinity assembly. Nr,Swiss-Prot,KEGG and COG/KOG databases were selected for annotation analysis. The differentially expressed genes(DEGs)were screened and their trends were analyzed on the Omicsmart platform. Then GO functional annotation analysis and KEGG signal pathway enrichment analysis were performed,and five DEGs were randomly selected for real-time quantitative PCR(qRT-PCR)validation.【Result】A total of 93227 uni genes whose range were from 201 to 35402 bp were obtained through transcriptome sequencing,with their average length of 834 bp and the N50 of 1352 bp. 4750 genes were identified as DEGs through pairwise comparison between groups,of which the up-regulated genes were 3557 and the down-regulated genes were 2829. More than fifty percent were significantly enriched in six gene expression trends(P<0.01):Profile 0 was enriched in 415 genes;Profile 5 was enriched in 201 genes; Profile 11 was enriched in 371 genes;Profile 13 was enriched in 841 genes;Profile 17 was enriched in 387 genes and Profile 18 was enriched in 411 genes.The DEGs in six trends were annotated to metabolic processes,cellular processes,single organism process,cell,cell parts,macromolecular complex and catalytic activity by GO functional analysis. The KEGG signaling pathway enrichment analysis showed that DEGs in Profile 13 were enriched in 86 pathways at most and significantly enriched in ribosome,carbon metabolism,oxidative phosphorylation,biosynthesis of amino acids,protein processing in endoplasmic reticulum,glycolysis or gluconeogenesis,glutathione metabolism and protein export.【Conclusion】The gill tissues of E.carinicauda synthesizes protein and increases metabolic capacity to resist hypoxia in the early stage of hypoxia stress. But with the prolongation of hypoxia,both substance synthesis and energy metabolism were affected and decreased. After reoxygenation,with the prolongation ofreoxygenation,the protein synthesis and energy metabolism gradually recover to level under normal oxygen.
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