Calmodulin (CaM) is a highly conserved second messenger protein transducing calcium signals by binding and modulating intracellular calcium ions (Ca²⁺), and involves in the Ca²⁺-dependent physical processes including host defense in vertebrates. In the present study, a CaM homologue (designated as CgCaM) was identified from Pacific oyster Crassostrea gigas. The open reading frame of CgCaM cDNA was of 471 bp encoding a polypeptide of 156 amino acid residues. There were four EFh domains predicted in CgCaM, which shared high homologies with those in CaMs from oyster C. virginica and other invertebrates. The mRNA transcripts of CgCaM were constitutively expressed in all the tested tissues including labellum, mantle, gonad, gills, adductor muscle, haemocytes and hepatopancreas, with the highest expression level in haemocytes. The mRNA expression level of CgCaM in haemocytes decreased significantly (0.31-fold of that in blank, p < 0.05) at 3 h after LPS stimulation, while the intracellular Ca²⁺ (1.57-fold of that in blank, p < 0.05) and the mRNA expression of cytokine CgIL17-1 (4.87-fold of that in blank, p < 0.05) both increased in haemocytes. Meanwhile, an oyster miRNA scaffold659_26519 was identified, and it was proved to target the 3′-untranslated regions (3′-UTR) of CgCaM mRNA by luciferase reporter assay. The expression of scaffold659_26519 increased significantly at 3 h (43.523-fold of that of blank, p < 0.05) and 6 h (55.91-fold of that of blank, p < 0.05) after LPS stimulation. When the expression of scaffold659_26519 was inhibited by transfection with its inhibitor in vitro, the expression of CgIL17-1 declined significantly to 0.58-fold of that in LPS stimulation group. These findings indicated that the miRNA scaffold659_26519 targeted CaM was involved in the early inflammatory response of oyster immunity, and provided a new evidence for CaM-mediated immune mechanism in molluscs.

钙调素（CaM）是一种高度保守的第二信使蛋白，通过结合和调节细胞内钙离子（Ca ²⁺）来传导钙信号，并参与脊椎动物体内包括宿主防御在内的Ca ²⁺依赖性物理过程。在本研究中，从太平洋牡蛎Crassostrea gigas中鉴定出一个 CaM 同源物（称为Cg CaM） 。Cg CaM cDNA的开放阅读框为471 bp，编码156个氨基酸残基的多肽。在Cg CaM中预测有四个 EFh 结构域，它们与来自牡蛎C. virginica和其他无脊椎动物的 CaM 具有高度同源性。mRNA转录本Cg CaM在所有受试组织中组成型表达，包括唇、外套膜、性腺、鳃、内收肌、血细胞和肝胰腺，在血细胞中表达水平最高。LPS刺激3 h后血细胞中Cg CaM mRNA表达水平显着降低（为空白的0.31倍，p  < 0.05），而细胞内Ca ²⁺（为空白的1.57倍，p  < 0.05）细胞因子Cg IL17-1的 mRNA 表达（是空白的 4.87 倍，p  < 0.05）在血细胞中均增加。同时，鉴定出牡蛎miRNA支架659_26519，并证明其靶向Cg的3'-非翻译区（3'-UTR）通过荧光素酶报告基因测定的 CaM mRNA。 在LPS刺激后3 h（空白的43.523倍，p  < 0.05）和6 h（空白的55.91倍， p < 0.05），scaffold659_26519的表达显着增加。体外转染支架659_26519的抑制剂后， Cg IL17-1的表达明显下降至LPS刺激组的0.58倍。这些发现表明，靶向CaM的miRNA支架659_26519参与了牡蛎免疫的早期炎症反应，为软体动物中CaM介导的免疫机制提供了新的证据。