高温下牙鲆鳃中ripk3基因表达与启动子甲基化的关联分析

Association of ripk3 Expression with Promoter Methylation in the Gill of Olive Flounder (Paralichthys olivaceus) under Heat Stress

摘要:

近年来，随着近岸海水水温的持续升高，高温已成为制约牙鲆（Paralichthys olivaceus）等经济鱼类健康养殖的重要因素。鳃是鱼类响应环境胁迫的“前沿哨所”，其结构与功能的完整性对维持机体生存至关重要。Ripk3 基因作为热应激下调控细胞死亡的关键因子，在调控鳃细胞死亡及损伤过程中发挥重要作用。为明确高温胁迫下牙鲆鳃中ripk3基因的表达水平与启动子甲基化水平的关系，本研究设置20℃对照组及 28 ℃、30 ℃、32 ℃ 三个梯度高温组，对 6 月龄、体重 73.87 ± 16.1 g 的牙鲆分别处理 0、2、4、8、12 h。通过 qRT-PCR 检测 ripk3 基因的表达水平，采用亚硫酸氢盐测序法（BSP）分析 ripk3 启动子的甲基化状态，并对基因表达水平与甲基化水平进行关联分析。结果显示，ripk3 基因的表达呈显著“温度-时间”双依赖激活特征，所有高温组的表达量均在胁迫 2 h 时达到峰值，且 30 ℃ 高温组的表达上调幅度最为明显。在 ripk3 启动子中的核心调控区域存在 7 个关键 CpG 位点（CpG19、CpG51、CpG74、CpG120、CpG142、CpG161、CpG182），其中 CpG74 为 30 ℃ 特异性调控位点，CpG142 和 CpG182 为 32 ℃ 核心靶点，且 CpG182 与 CpG120、CpG142、CpG161 呈高强度正相关，构成核心协同调控模块。结果还表明 ripk3 基因启动子的整体及单个位点的甲基化与基因表达的关联具有显著的温度特异性，在启动子高甲基化状态下的 28 ℃ 组中，ripk3 基因的表达有部分被激活；在 30 ℃ - 高温胁迫的临界耐受温度组中，ripk3 基因表达上调的幅度是最明显的，甲基化水平与基因表达的关联关系呈“甲基化下降-表达上调”经典 DNA 甲基化调控模式，且 CpG74 甲基化水平与基因表达呈强正相关（r = 0.5016，p ＜ 0.05）；在 32 ℃ 组中，该关联呈中等正相关。因此，ripk3 基因启动子关键位点的去甲基化，可能在高温诱导其表达的调控过程中发挥重要作用。本研究的结果可为深入探究鱼类响应高温胁迫的分子机制提供新的思路。

Abstract:

In recent years, with the continuous rise of inshore seawater temperature, high temperature has become an important limiting factor for the healthy aquaculture of economic fish such as olive flounder (Paralichthys olivaceus). The gill serves as a "frontline outpost" for fish in response to environmental stress, and the integrity of its structure and function directly determines the survival ability of fish. As a key regulatory gene that induces cell death (including necroptosis) under heat stress, the receptor-interacting serine/threonine kinase 3 (ripk3) gene plays an important role in the regulation of gill injury process. However, the expression pattern of ripk3 gene in olive flounder under high temperature stress, especially the relationship between its expression and promoter methylation, remains unclear. This study aimed to clarify the ripk3 gene expression, and its association with promoter methylation in the gills of olive flounder under different high-temperature. The results will reveal the epigenetic regulatory mechanism of the ripk3 gene in response to high-temperature stress and provide new insights for research on fish thermotolerance. The experimental subjects were 6-month-old olive flounder with an average body weight of 73.87±16.1 g. The experiment set up a control group at 20℃ (the optimal growth temperature for olive flounder) and three gradient high-temperature groups at 28℃, 30℃, and 32℃. The fish in each group were subjected to high-temperature stress for 0 h, 2 h, 4 h, 8 h, and 12 h, respectively. Three biological replicates were set for each temperature group. Gills from 5 fish were sampled in each temperature group. The expression level of ripk3 gene was detected by quantitative real-time polymerase chain reaction (qRT-PCR). Three technical replicates were set for each sample to ensure the reliability of the experimental data. The relative mRNA expression levels of the genes were calculated using the 2?ΔΔCt method. The promoter methylation status of ripk3 gene was analyzed by bisulfite sequencing PCR (BSP). At least 10 valid clones per sample were used and the methylation rate of each CpG site was calculated as number of methylated clones / total number of valid clones × 100%. The overall or site-specific methylation level was then determined. The experimental data were statistically analyzed using Pearson correlation analysis was used to clarify the correlation between ripk3 gene expression and promoter methylation level. The results of qRT?PCR showed that ripk3 gene expression in the gills of olive flounder displayed a significant temperature? and time?dependent activation pattern. In the 20℃ control group, ripk3 transcript levels remained low and stable throughout the experiment, with no significant differences among stress time points (p>0.05). At all stress time points, the ripk3 expression levels in all three high-temperature groups (28℃, 30℃, 32℃) were significantly higher than those in the control group (p<0.05). Specifically, the ripk3 expression level of the ripk3 gene in all high?temperature groups increased rapidly at the beginning of heat treatment, peaked at 2 h, and then gradually decreased with prolonged treatment time. Nevertheless, it remained significantly higher than that in the control group at 12 h. Among the three high-temperature groups, the most significant up-regulation of ripk3 gene was observed in the 30℃ group, and its peak expression at 2 h was significantly higher than that in the 28℃ and 32℃ groups. These results indicated that 30℃ might be the critical tolerance temperature for olive flounder under high temperature stress. BSP sequencing results showed that there were 7 key CpG sites (CpG19, CpG51, CpG74, CpG120, CpG142, CpG161, CpG182) in the core regulatory region of the ripk3 gene promoter in olive flounder, which were closely associated with the regulation of ripk3 gene expression. Further analysis found that the methylation levels and regulatory mode of these 7 key CpG sites exhibited significant temperature specificity. Among them, CpG74 was a 30℃-specific regulatory site, whose methylation level was significantly positively correlated with ripk3 expression (r=0.5016, p<0.05). The CpG142 and CpG182 sites were the core target sites at 32℃, and their methylation levels were significantly positively correlated with ripk3 gene expression. In addition, correlation analysis among the 7 key CpG sites showed that CpG182 was strongly positively correlated with CpG120, CpG142, and CpG161 (all p<0.05), forming a core synergistic regulatory module that might jointly participate in the regulating ripk3 gene expression under high- temperature stress. The relationship between the overall methylation level of the ripk3 gene promoter and its expression exhibited significant temperature specificity, which differed from the traditional simple negative correlation model. In the 28℃ group, although the overall methylation level of the ripk3 gene promoter was relatively high, the ripk3 gene expression was still partially activated, showing a weak positive correlation between promoter methylation and gene expression (p>0.05). In the 30℃ group (the critical tolerance temperature for olive flounder in response high-temperature stress), the overall methylation level of the ripk3 gene promoter was significantly lower than that in the other two high-temperature groups. Meanwhile, a significant negative correlation between promoter methylation and gene expression was observed, displaying a classic DNA methylation regulatory pattern of “hypomethylation accompanied by upregulated expression”. In the 32℃ group, there was a moderate positive correlation between the overall methylation level of the promoter and the ripk3 gene expression (p<0.05). In conclusion, this study systematically investigated the expression pattern of ripk3 gene and the methylation status of its promoter in the gills of olive flounder under different high temperature stresses. The results demonstrated that the ripk3 gene expression showed a significant temperature- and time- dependent activation characteristic, and the most significant up-regulation of expression presented in the 30℃ group. The seven key CpG sites in the core regulatory region of the ripk3 promoter displayed temperature-specific methylation regulatory characteristics, and the demethylation of these key CpG sites might be the core mechanism underlying high temperature-induced ripk3 gene expression. Among them, CpG74, as a 30℃-specific regulatory site, played a key role in the significant up-regulation of ripk3 gene expression under 30℃ stress. These findings not only clarify the regulatory relationship between ripk3 gene expression and promoter methylation in olive flounder under high temperature stress, but also reveal the epigenetic regulatory mechanism of fish in response to high temperature stress from a new perspective. This study provides new insights into the mechanism of fish response to high-temperature stress and theoretical support for the healthy aquaculture of olive flounder in the context of global warming.