Evaluating the Hypoxia Response of Ruffe and Flounder Gills by a Combined Proteome and Transcriptome Approach

Hypoxia has gained ecological importance during the last decades, and it is the most dramatically increasing environmental factor in coastal areas and estuaries. The gills of fish are the prime target of hypoxia and other stresses. Here we have studied the impact of the exposure to hypoxia (1.5 mg O2/l for 48 h) on the protein expression of the gills of two estuarine fish species, the ruffe (Gymnocephalus cernua) and the European flounder (Platichthys flesus). First, we obtained the transcriptomes of mixed tissues (gills, heart and brain) from both species by Illumina next-generation sequencing. Then, the gill proteomes were investigated using two-dimensional gel electrophoresis and mass spectrometry. Quantification of the normalized proteome maps resulted in a total of 148 spots in the ruffe, of which 28 (18.8%) were significantly regulated (> 1.5-fold). In the flounder, 121 spots were found, of which 27 (22.3%) proteins were significantly regulated. The transcriptomes were used for the identification of these proteins, which was successful for 15 proteins of the ruffe and 14 of the flounder. The ruffe transcriptome dataset comprised 87,169,850 reads, resulting in an assembly of 72,108 contigs (N50 = 1,828 bp). 20,860 contigs (26.93%) had blastx hits with E < 1e-5 in the human sequences in the RefSeq database, representing 14,771 unique accession numbers. The flounder transcriptome with 78,943,030 reads assembled into 49,241 contigs (N50 = 2,106 bp). 20,127 contigs (40.87%) had a hit with human proteins, corresponding to 14,455 unique accession numbers. The regulation of selected genes was confirmed by quantitative real-time RT-PCR. Most of the regulated proteins that were identified by this approach function in the energy metabolism, while others are involved in the immune response, cell signalling and the cytoskeleton.

近数十年来，低氧（Hypoxia）在生态学领域的重要性日益凸显，且已成为沿海区域与河口生境中增长最为显著的环境胁迫因子。鱼类鳃部是低氧及其他环境胁迫的首要作用靶标。本研究针对两种河口鱼类——梅花鲈（Gymnocephalus cernua）与欧洲鲽（Platichthys flesus）——开展实验，探究了低氧暴露（1.5 mg O₂/L，持续48小时）对其鳃组织蛋白质表达的影响。首先，本研究通过Illumina二代测序（Illumina next-generation sequencing）技术，获取了两种鱼类的混合组织（鳃、心脏与脑组织）转录组数据。随后，采用二维凝胶电泳（two-dimensional gel electrophoresis）与质谱（mass spectrometry）联用技术，对其鳃组织蛋白质组进行分析。对标准化后的蛋白质组图谱进行定量分析，结果显示梅花鲈样本中共检出148个蛋白斑点，其中28个（占比18.8%）的表达量存在显著变化（变化倍数>1.5倍）；欧洲鲽样本中共检出121个蛋白斑点，其中27个（占比22.3%）的蛋白质表达量存在显著调控变化。本研究利用转录组数据对上述差异表达蛋白进行鉴定，其中梅花鲈成功鉴定出15种蛋白，欧洲鲽成功鉴定出14种蛋白。梅花鲈的转录组原始数据包含87,169,850条测序读段（reads），经组装后得到72,108条重叠群（contig），其N50值为1828 bp；其中20,860条重叠群（占比26.93%）在参考序列数据库（RefSeq database）的人类蛋白序列中通过BLASTx比对获得显著匹配（E值<1e-5），对应14,771个唯一登录号。欧洲鲽的转录组原始数据包含78,943,030条测序读段（reads），经组装后得到49,241条重叠群，其N50值为2106 bp；其中20,127条重叠群（占比40.87%）可与人类蛋白序列匹配，对应14,455个唯一登录号。本研究通过实时定量反转录PCR（quantitative real-time RT-PCR）验证了部分筛选基因的表达调控情况。经本方法鉴定得到的差异表达蛋白中，多数参与能量代谢过程，其余则分别与免疫应答、细胞信号传导及细胞骨架功能相关。