setd7基因编辑提高尼罗罗非鱼的低氧耐受能力

Exploration of enhancing hypoxia tolerance in Nile tilapia (Oreochromis niloticus) through setd7 gene editing

  • 摘要:
    目的 探究甲基转移酶基因setd7在鱼类低氧耐受中的功能,以尼罗罗非鱼为实验模型,通过基因编辑技术构建setd7基因突变品系,评估其在低氧环境下的生理响应及耐受能力,为鱼类耐低氧分子育种及甲基转移酶功能机制研究提供理论依据和实验材料。
    方法 利用CRISPR/Cas9基因编辑技术,针对尼罗罗非鱼setd7基因第2个外显子的氨基酸保守区域设计特异性敲除靶点,并对一细胞期受精卵进行显微注射。通过T7E1酶切实验和测序分析验证基因编辑效率,成功获得−23 bp碱基缺失的setd7+/−突变品系。进一步对F1的1月龄罗非鱼进行低氧胁迫处理,观察其缺氧表征及存活时间,评估其低氧耐受能力。
    结果 setd7+/−突变品系由于移码突变导致SETD7蛋白功能丧失,setd7基因表达量较野生型显著降低。在低氧胁迫实验中,setd7+/−突变体尼罗罗非鱼表现出更强的低氧耐受能力,其浮头现象出现时间显著晚于野生型,且在低氧环境下的存活时间明显延长。这表明setd7基因突变显著增强了尼罗罗非鱼的低氧适应能力。
    结论 甲基转移酶基因setd7的突变能够显著提高尼罗罗非鱼的低氧耐受能力,为耐低氧尼罗罗非鱼分子育种提供了重要的实验材料和理论基础。同时,该研究为进一步探索甲基转移酶在低氧适应中的分子机制及功能研究奠定了科学基础。

     

    Abstract: This study aims to explore the function of the methyltransferase gene setd7 in hypoxia tolerance in fish, using Nile tilapia (Oreochromis niloticus) as the experimental model. By employing gene editing technology, we constructed setd7 gene mutation lines to assess their physiological responses and tolerance capabilities in low oxygen environments, providing theoretical basis and experimental materials for molecular breeding of hypoxia tolerant fish and research on the functional mechanisms of methyltransferases. Using CRISPR/Cas9 gene editing technology, specific knockout targets were designed for the conserved amino acid region of the second exon of the setd7 gene in O. niloticus, and microinjection was performed on one cell fertilized eggs. The gene editing efficiency was verified through T7E1 enzyme digestion experiments and sequencing analysis, successfully obtaining the setd7+/− mutation line with a -23 bp base deletion. Further, one month old F1 generation tilapia were subjected to hypoxic stress treatment to observe their hypoxia characteristics and survival time, assessing their hypoxia tolerance. The experimental results showed that the setd7+/− mutation line exhibited a loss of function of the SETD7 protein due to a frameshift mutation, with a significantly reduced expression level of the setd7 gene compared to the wild type. In the hypoxic stress experiment, the setd7+/− mutant tilapia demonstrated stronger hypoxia tolerance, with the onset of surface swimming occurring significantly later than in the wild type, and a markedly extended survival time in low oxygen environments. This indicates that the mutation of the setd7 gene significantly enhances the hypoxia adaptation ability of tilapia. This study found that mutations in the methyltransferase gene setd7 can significantly improve the hypoxia tolerance of Nile tilapia, providing important experimental materials and theoretical foundations for molecular breeding of hypoxia tolerant tilapia. Additionally, this research lays a scientific foundation for further exploration of the molecular mechanisms and functional studies of methyltransferases in hypoxia adaptation.

     

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