水中的溶解氧(Dissolved Oxygen, DO)水平影响着鱼类的生存、繁殖、生长和发育。缺氧会对水产养殖业的发展,生物多样性以及生态系统的功能产生影响。因此,本研究以红鳍东方鲀(Takifugu ru-bripes)为研究对象,对常氧(7.5 ± 0.5 mg/L)与低氧(2.5 ± 0.5 mg/L,10天)环境下的红鳍东方鲀的鳔组织进行了转录组测序分析,以探究其应对低氧胁迫时的分子调控机制。随之,筛选出差异表达基因并对其进行生物信息学分析。同时,进一步采用qRT-PCR进行了验证。结果显示,在红鳍东方鲀的鳔组织中共鉴定到了536个差异基因,包括493个上调基因和43个下调基因。KEGG富集结果表明,钙信号传导途径,环磷酸腺苷(cAMP)通路,胰岛素分泌三个信号通路通过调整机体内离子转运,在机体适应低氧环境、维持细胞存活中发挥了重要作用。此外,GSEA富集分析表明,红鳍东方鲀还通过下调DNA复制,细胞周期等耗能的生物过程来抑制细胞正常分裂和生长,从而减少低氧环境中的能量消耗。最后,我们随机挑选了转录组数据中的6个基因进行qRT-PCR验证,结果与转录组数据分析相一致,证实了转录组测序结果的可靠性。本研究结果不仅丰富了红鳍东方鲀响应低氧胁迫的分子调控机制,同时为探究鱼类的低氧适应机制提供了理论依据。
Dissolved oxygen (DO) in water affects the fish’s survival, reproduction, growth, and development. Hypoxia can significantly impact the development of aquaculture, biodiversity, and ecosystem function. Therefore, in this study, the transcriptome of Takifugu rubripes was sequenced to investigate the molecular regulatory mecha-nisms involved in the response to chronic hypoxic stress in the swimbladder under normoxic (7.5 ± 0.5 mg/L) and hypoxic (2.5 ± 0.5 mg/L, 10 days) conditions. Concomitantly, differentially expressed genes (DEGs) were identified and bioinformatics analysis of the DEGs was performed. At the same time, qRT-PCR validation was also carried out. The results showed that a total of 536 DEGs were identified in the T. rubripes swimbladder, including 493 up-regulated genes and 43 down-regulated genes. According to the KEGG enrichment analysis, the calcium signaling pathway, cAMP signaling pathway, and insulin secretion played an important role in adapting to the hypoxic environment and maintaining cell survival by regulating ion transport in the organism. In addition, GSEA en-richment analysis showed that T. rubripes could inhibit cell division and growth via down-regulating energy-consuming biological processes to reduce energy consumption in hypoxic environments, including DNA replication and cell cycle. Finally, 6 genes were randomly selected for qRT-PCR validation and the results proved to be consistent with the transcriptome data analysis, which confirmed the reliability of the transcriptome data. The results of this study not only en-riched the molecular regulatory mechanism of T. rubripes in response to hypoxic stress, but also provided a theoretical basis for exploring the hypoxia adaptation mechanism of fish.
Dissolved oxygen (DO) in water affects the fish’s survival, reproduction, growth, and development. Hypoxia can significantly impact the development of aquaculture, biodiversity, and ecosystem function. Therefore, in this study, the transcriptome of Takifugu rubripes was sequenced to investigate the molecular regulatory mechanisms involved in the response to chronic hypoxic stress in the swimbladder under normoxic (7.5 ± 0.5 mg/L) and hypoxic (2.5 ± 0.5 mg/L, 10 days) conditions. Concomitantly, differentially expressed genes (DEGs) were identified and bioinformatics analysis of the DEGs was performed. At the same time, qRT-PCR validation was also carried out. The results showed that a total of 536 DEGs were identified in the T. rubripes swimbladder, including 493 up-regulated genes and 43 down-regulated genes. According to the KEGG enrichment analysis, the calcium signaling pathway, cAMP signaling pathway, and insulin secretion played an important role in adapting to the hypoxic environment and maintaining cell survival by regulating ion transport in the organism. In addition, GSEA enrichment analysis showed that T. rubripes could inhibit cell division and growth via down-regulating energy-consuming biological processes to reduce energy consumption in hypoxic environments, including DNA replication and cell cycle. Finally, 6 genes were randomly selected for qRT-PCR validation and the results proved to be consistent with the transcriptome data analysis, which confirmed the reliability of the transcriptome data. The results of this study not only enriched the molecular regulatory mechanism of T. rubripes in response to hypoxic stress, but also provided a theoretical basis for exploring the hypoxia adaptation mechanism of fish.
将每组的红鳍东方鲀的鳔组织分别置于液氮中进行研磨,研磨后的样品使用Trizol试剂(Invitrogen, Carlsbad, CA)进行总RNA的提取。RNA提取完成后,使用1%琼脂糖凝胶电泳检测分析RNA的完整性以及RNA是否存在DNA的污染,用NanoPhotometer分光光度计(IMPLEN, California, CA, USA)来检测RNA的纯度,并使用Agilent 2100生物分析仪(Agilent Technologies, Santa Clara, CA, USA)来精确检测RNA的纯度和完整性。
在红鳍东方鲀的样本质量检测合格后,选取常氧组(CSB1, CSB2, CSB3)和低氧组(ESB1, ESB2, ESB3)的鳔组织各3个样本(每个样本有4个生物学重复),共6个样本进行文库的构建和测序,本实验委托诺禾致源公司进行测序。再按照NEB普通建库方式进行cDNA文库的构建,建库试剂盒为NEBNext®UltraTM RNA Library Prep Kit (Illumina,美国),所有的文库制备步骤均按照试剂盒说明书进行:首先通过磁珠富集带有polyA尾的mRNA,在逆转录酶体系中合成单链cDNA。然后以dNTPs为原料,在DNA聚合酶I系统中再合成双链cDNA。双链cDNA经过末端修复后,加入polyA尾和测序接头到文库中。用AMPure XP beads筛选250~300 bp左右的cDNA,进行PCR扩增并再次使用AMPure XP beads纯化PCR产物,最终获得文库。在本次实验中共构建了6个RNA (cDNA)文库(包括3个常氧组和3个缺氧组,每组包含4个生物重复),用于后续RNA测序和生物信息学分析。在cDNA文库构建完成后,所有的cDNA文库均在Illumina Novaseq 6000平台(Illumina,美国)上进行测序。
除了KEGG富集结果外,离子运输相关条目在GO聚类分析中也显著富集,包括钠离子运输(Sodium Ion Transport),钠离子跨膜运输(Sodium Ion Transmembrane Transport),钾离子跨膜运输(Potassium Ion Transmembrane Transport),钾离子运输(Potassium Ion Transport)。且这些条目能够被很好地聚类到“钾离子进入细胞”(Potassium Ion Import Across)和“钠离子进入细胞”(Sodium Import into Cell)这两个大类中。大量离子转运相关基因在这些通路中富集,这与我们的KEGG结果一致。
陆 芸,李 岩,尚凤芹,韩 冰,魏仁杰,王秀利,刘 洋. 长期低氧胁迫下红鳍东方鲀鳔的转录组分析 Transcriptome Analysis of the Takifugu rubripes Swimbladder under Chronic Hypoxic Stress[J]. 海洋科学前沿, 2023, 10(01): 5-21. https://doi.org/10.12677/AMS.2023.101002
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