三角帆蚌IGF2BP通过AKT通路促进外套膜矿化相关研究

PROMOTION OF MANTLE MINERALIZATION BY AKT MEDIATED IGF2BP IN HYRIOPSIS CUMINGII

  • 摘要: 为研究胰岛素样生长因子结合蛋白(Insulin-like growth factor binding protein, IGFBP)对贝类生物矿化的调控功能, 克隆三角帆蚌(Hyriopsis cumingii)IGF2BP基因, 构建其插核后组织表达谱, 采用活体注射技术研究插核育珠后三角帆蚌外源添加人源IGF2BP2和抑制IGF2BP2 (CWI1-2)处理, 对AKT信号通路介导的胰岛素样生长因子1受体(IGF1R)及矿化相关基因几丁质酶(CHS1)和骨形态发生蛋白10 (BMP10)表达的影响, 并通过组织切片染色分析干预后插核部位组织形态变化。研究结果表明, 三角帆蚌IGF2BP基因CDS区1824 bp, 编码607AA, 含6个结构域, 符合IGF2BPs典型的保守结构域特征, 蛋白相对分子量为66.69 kD, 理论等电点为8.96; 而插核育珠能显著提高HcIGF2BP mRNA表达水平, 且在插核后各时间点上呈现显著性的先上升后缓慢下降的趋势, 在21d时其表达量最高(P<0.05); 外源添加IGF2BP2在14d时能够显著促进HcIGF2BPCHS1IGF1R的表达(P<0.05), 同时, 在14d和28d显著促进AKT1BMP10的表达(P<0.05), 而抑制组在14d时显著抑制HcIGF2BPCHS1IGF1RAKT1的基因表达(P<0.05), 在14d和28d时显著抑制下游基因BMP10的表达(P<0.05)。组织切片观察结果表明外源IGF2BP2促进插核部位细胞在珠核表面进行迁移并增殖, 促进细胞形态转变。研究表明三角帆蚌IGF2BPs能够通过AKT信号通路调控珍珠形成相关关键矿化基因表达, 影响珍珠囊结构, 为进一步探究IGF系统在贝类矿化功能中的调控作用奠定理论基础, 为加速珍珠培育进程、缩短培育时间提供分子依据。

     

    Abstract: Insulin-like growth factor binding protein (IGFBP) plays an important role in various biological processes such as cell growth, apoptosis, bone formation by binding IGF. Additionally, it exerts regulatory functions on shellfish biomineralization. In this study, the IGF2BP gene was cloned for the first time, and its tissue expression profile was constructed after nucleation. Subsequently, human IGF2BP2 was introduced after nucleation, resulting in the suppression of IGF2BP2 (CWI1-2). The effects on the expression of insulin-like growth factor 1 receptor (IGF1R) mediated by AKT signaling pathway and the mineralization related genes chitinase (CHS1) and bone morphogenetic protein 10 (BMP10) were analyzed through tissue section staining. The results showed that the CDS region of IGF2BP gene was 1824 bp, encoding 607 Aa and containing 6 domains, consistent with the typical conserved domain characteristics of IGF2BPs. The relative molecular weight of the protein was 66.69 kDa, with a theoretical isoelectric point of 8.96. The expression level of HcIGF2BP mRNA increased significantly after nucleation, exhibiting a notable trend of initial elevation followed by a gradual decline at each time point after nucleation, peaking at 21d (P<0.05); Exogenous addition of IGF2BP2 can significantly promote the expression of HcIGF2BP, CHS1, and IGF1R at the 14th day (P<0.05), and significantly promoted the expression of AKT1 and BMP10 on the 14th and 28th days (P<0.05). In the inhibition group, the gene expression of HcIGF2BP, CHS1, IGF1R, and AKT1 was significantly suppressed on the 14th day (P<0.05), leading to a significantly inhibition of downstream gene BMP10 expression on the 14th and 28th days (P<0.05). Tissue section observation results showed that exogenous IGF2BP2 promoted cell migration and proliferation on the surface of nucleus nucellus, facilitating cell morphological transformation. The results of this study indicate that IGF2BPs can regulate the expression of key mineralization genes relevant to pearl formation through the AKT signaling pathway, influencing the structure of pearl sac. This study lays a theoretical basis for further exploring the regulatory role of IGF system in the mineralization function of shellfish, and providing molecular basis for accelerating the pearl cultivation process and shortening the cultivation time.

     

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