马氏珠母贝IGF2BP1基因的鉴定及对矿化相关基因表达的影响

Identification of IGF2BP1 and its effect on the expression of mineralization-related genes in Pinctada fucata martensii

  • 摘要: 为了研究胰岛素样生长因子2结合蛋白(IGF2BPs)基因是否参与珍珠贝的生物矿化,本实验鉴定了一个马氏珠母贝IGF2BP1基因,命名为PfIGF2BP1-1,该基因cDNA全长为7 348 bp,ORF长 1 818 bp,编码605个氨基酸。多序列比对和系统进化分析表明,PfIGF2BP1-1有2个RRM结构域和4个KH结构域,与其他物种来源的IGF2BPs同源。实时荧光定量PCR结果显示,PfIGF2BP1-1在马氏珠母贝的8个组织中均有表达,在闭壳肌组织中表达量最高,其次为足和外套膜组织。利用pET-32a 原核表达载体构建了含有PfIGF2BP1-1成熟多肽区的重组质粒并成功表达蛋白。在IPTG浓度为0.5 mmol/L,37 ℃培养8 h的条件下诱导表达PfIGF2BP1-1蛋白最佳。PfIGF2BP1-1蛋白刺激马氏珠母贝外套膜原代细胞,引起壳基质蛋白基因AccbpMSI7、Nacrein的表达水平显著上升,表明PfIGF2BP1-1蛋白可诱导壳矿化相关基因的表达,参与马氏珠母贝的生物矿化过程。

     

    Abstract: Insulin-like growth factor 2 binding proteins (IGF2BPs) are the important RNA metabolism regulators involved in many different biological processes. In order to study whether IGF2BP1 plays a role in biomineralization of pearl oysters, this study identified a Pinctada fucata martensii IGF2BP1 gene, named PfIGF2BP1-1. The cDNA length of the gene is 7 348 bp, with an ORF of 1 818 bp and encoding 605 amino acids. Multiple sequence alignments and phylogenetic analysis showed that PfIGF2BP1-1 has two RRM domains and four KH domains, which are homologous to IGF2BPs from other species. Real-time fluorescent quantitative PCR results showed that PfIGF2BP1-1 was expressed in eight tissues of P. fucata martensii, with the highest expression level in adductor muscle tissue, followed by foot and mantle tissue. The pET-32a prokaryotic expression vector was used to construct a recombinant plasmid containing the mature polypeptide region of PfIGF2BP1-1 and the protein was successfully expressed. The optimal conditions for inducing protein expression were that the bacteria were cultured at 37 ℃ for 8 hours with 0.5 mmol/L IPTG . The PfIGF2BP1-1 protein can stimulate the coat membrane primary cells of P. fucata martensii and significantly increase the expression level of cell matrix protein genes Accbp, MSI7, and Nacrein in primary mantle cells. The results indicated that the PfIGF2BP1-1 protein is involved in the biomineralization process of P. fucata martensii through inducing the expression of shell mineralization-related genes.

     

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