Effects of hdac8 knockout on low temperature tolerance in zebrafish (Danio rerio)
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摘要: 为探究敲除组蛋白去乙酰化酶8基因 (histone deacetylase 8, hdac8) 对斑马鱼(Danio rerio)低温耐受能力的影响,采用组织学、生理和生化等方法,研究了低温(8 ℃) 短期胁迫下hdac8基因敲除(hdac8-/-)斑马鱼和野生型(WT)斑马鱼的组织损伤、氧化应激相关指标及其凋亡相关基因表达的变化。结果表明:低温胁迫下,hdac8-/-斑马鱼的半数致死时间(LT50)明显缩短,hdac8-/-斑马鱼的LT50为14.5 h,WT斑马鱼的LT50为21.5 h;HE染色显示,低温胁迫下,hdac8-/-斑马鱼鳃丝、肝脏和骨骼肌相较于WT斑马鱼损伤更加严重;28 ℃下,WT和hdac8-/-斑马鱼的活性氧(ROS)、丙二醛(MDA)水平无显著性变化(P>0.05),而hdac8-/-斑马鱼的ATP水平则显著降低(P<0.05);低温胁迫下,hdac8-/-斑马鱼的 ROS和MDA水平显著高于WT斑马鱼(P<0.05),而ATP水平则显著低于WT斑马鱼(P<0.05);RT-qPCR显示,28 ℃下, hdac8-/-斑马鱼caspase3基因表达显著上调(P<0.05),其他抗氧化与凋亡相关基因无显著性变化(P>0.05);低温胁迫下,WT和hdac8-/-斑马鱼sod、cat、caspase3、caspase9和bax基因表达水平显著上调(P<0.05),且hdac8-/-斑马鱼的这5种基因表达水平均显著高于WT斑马鱼(P<0.05)。研究表明,敲除hdac8基因显著降低了斑马鱼的低温耐受能力,并促进了低温氧化应激损伤和细胞凋亡。
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关键词:
- 斑马鱼 /
- 组蛋白去乙酰化酶8基因 /
- 低温耐受 /
- 氧化损伤 /
- 细胞凋亡
Abstract: To explore the effects of knocking out histone deacetylase 8 gene (hdac8) on low-temperature tolerance of zebrafish (Danio rerio), the changes in tissue damage of gill filaments, liver, and skeletal muscles, oxidative stress-related indices, and apoptosis-related gene (caspase3, caspase9, bcl-2, bax and bcl-2/bax) expression were detected in 8 months old hdac8 deficient (hdac8-/-) and wild type (WT) zebrafish exposed to low water-temperature of 8℃ stress from 28℃ to 18℃ at a rate of 1℃/h and then to 8℃ at a rate of 0.5℃/h by histological, physiological and biochemical methods and gene expression analysis. The results showed that the median lethal time (LT50) was significantly decreased under low temperature stress in hdac8-/- zebrafish, with the LT50 of 14.5 h relative to the LT50 of 21.5 h in WT zebrafish. HE staining showed that more severe damage was observed in the gill filaments, liver and skeletal muscle in hdac8-/- zebrafish compared with WT zebrafish under low temperature stress. The ATP level in hdac8-/- zebrafish showed very significant decrease (P<0.05) at 28℃, without significant difference in the levels of reactive oxygen species (ROS) and malondialdehyde content between WT and hdac8-/- zebrafish (P>0.05). Under low-temperature stress, however, the hdac8-/- zebrafish had very significantly higher ROS and MDA levels than the WT zebrafish did (P<0.05), and very significantly lower ATP level than the WT zebrafish (P<0.05) did. RT-qPCR revealed that there was significantly up-regulated expression level of caspase3 in hdac8-/- zebrafish (P<0.05) at 28℃, without significant difference in the expression levels of other antioxidant and apoptosis-related genes (P>0.05). Under low-temperature stress, the expression levels of sod, cat, caspase3, caspase9 and bax were found to be significantly up-regulated in both WT and hdac8-/- zebrafish (P<0.05), significantly higher expression levels of these genes in hdac8-/- zebrafish than those in WT zebrafish (P<0.05). In conclusion, hdac8 knockout significantly impaired the tolerance to low temperature of zebrafish, and promoted oxidative damage and cell apoptosis at low temperature.-
Key words:
- Danio rerio /
- hdac8 /
- low-temperature tolerance /
- oxidative damage /
- cell apoptosis
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