卵形鲳鲹G蛋白偶联受体43基因克隆及营养调控分析

    Cloning and nutritional regulation analysis of the G-protein-coupled receptor 43 (GPR43) gene in Trachinotus ovatus

    • 摘要:
      背景 卵形鲳鲹(Trachinotus ovatus)是一种热带−亚热带海水鱼。近年来,中国卵形鲳鲹养殖业快速发展,其繁养殖关键技术获得突破,但随着养殖规模逐渐扩大,饲料需求增多,优化饲料配方降低养殖成本已成为提升卵形鲳鲹养殖收益的重要途径。
      目的 本实验探讨卵形鲳鲹G蛋白偶联受体43(GPR43)基因在其能量代谢中的作用。
      方法 根据卵形鲳鲹GPR43基因序列设计引物,鉴定出GPR43基因同源物,克隆GPR43基因编码区序列(CDS),利用生物信息学分析技术对该基因及其所编码的蛋白质结构特征进行分析;通过体内与体外实验,并采用实时荧光定量聚合酶链式反应(qRT-PCR)技术检测GPR43基因在卵形鲳鲹的10个不同组织中的表达水平;通过体内实验对卵形鲳鲹进行高糖饲喂和饥饿处理后,检测其对肝脏和肌肉中该基因表达水平的影响;在体外实验中,通过分离卵形鲳鲹肝脏原代细胞,分别进行高糖与饥饿处理后,再检测其对GPR43基因表达水平的影响。
      结果 卵形鲳鲹GPR43基因CDS为1 011 bp,编码336个氨基酸,是一种特异性识别短链脂肪酸的受体。蛋白质结构分析结果表明,卵形鲳鲹GPR43蛋白具有7个跨膜结构域;同源性分析结果显示,卵形鲳鲹GPR43基因与同物种的GPR43基因高度同源(氨基酸相似度在86%以上),并与阿纳鲳鲹(T. anakGPR43基因的进化关系最近,与哺乳动物同源性较低(氨基酸相似度为50%);检测组织分布结果显示,GPR43基因在卵形鲳鲹的肝脏中表达量最高,其次为肠道、脑和肌肉,在皮肤中表达最低;体内实验结果表明,高糖饲料饲喂的卵形鲳鲹的肝脏和肌肉组织中GPR43基因的表达水平整体上比饥饿组低;体外实验结果表明,肝脏原代细胞经过高糖处理后,其GPR43基因表达水平与饥饿组相比整体较低,趋势与体外实验较一致。
      结论 本研究结果表明,饲料中糖类水平显著调节了卵形鲳鲹肝脏和肌肉中GPR43基因的表达水平。这些发现提示该基因可能在感知能量状态和调节脂质代谢中发挥关键作用,在卵形鲳鲹养殖业降低饲养成本、控制鱼体脂肪含量和优化鱼肉品质等方面具有重要的参考意义。

       

      Abstract:
      Background Trachinotus ovatus is a tropical-subtropical marine fish species. In recent years, the aquaculture industry of T. ovatus in China has developed rapidly, with key breakthroughs in breeding and cultivation technologies. However, With the expansion of aquaculture scale and the subsequent increase in feed demand, optimizing feed formulations to reduce production costs has become a crucial approach for enhancing economic returns in aquaculture.
      Objective This study investigated the role of G-protein-coupled receptor 43 (GPR43) gene in energy metabolism in T. ovatus.
      Methods Based on the GPR43 gene sequence, primers were designed to identify homologs and clone the coding sequence (CDS). Bioinformatics analysis was subsequently employed to investigate the structural characteristics of the gene and its encoded protein. To further elucidate the functional roles, both in vivo and in vitro experiments were conducted. In vivo, the expression levels of GPR43 in ten different tissues of T. ovatus were detected using quantitative real-time polymerase chain reaction (qRT-PCR). Furthermore, the effects of high-sugar feeding and starvation on the expression of this gene in liver and muscle tissues were analyzed. In vitro studies involved isolating primary hepatocytes from T. ovatus liver, which were then subjected to high-sugar and starvation treatments to assess their impact on gene expression.
      Results The CDS of T. ovatus GPR43 gene was 1011 bp, encoding 336 amino acids, and it functions as a receptor specifically recognizing short-chain fatty acids. Protein structure analysis revealed that T. ovatus GPR43 trotein contains seven transmembrane domains. Homology analysis indicated that T. ovatus GPR43gene shared high homology with GPR43 gene of the same species (amino acid similarity>86%) and was most closely related to T. anak. In contrast, its homology with mammals was relatively low (amino acid similarity was 50%). Tissue distribution analysis showed that GPR43 gene expression was the highest in the liver, followed by the intestine, brain, and muscle, and the lowest in the skin. In vivo results demonstrated that GPR43 gene expression in the liver and muscle of fish fed a high-glucose diet was generally lower than that in the starvation group. Similarly, in vitro experiments showed that GPR43 gene expression in primary hepatocytes treated with high glucose was lower compared to the starvation group, consistent with the in vivo trend.
      Conclusion The results demonstrate that the expression levels of GPR43 gene in the liver and the muscle of T. ovatus are significantly modulated by dietary carbohydrate levels. These findings suggest that GPR43 gene plays a crucial role in sensing energy status and regulating lipid metabolism, thereby serving as a potential biomarker for energy homeostasis in this species. The present study provides significant reference value for reducing feeding costs, controlling body fat content, and improving flesh quality in T. ovatus aquaculture.

       

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