Identification of calmodulin (ScCaM) gene and its correlation with shell calcium carbonate deposition of Sinonovacula constricta
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摘要: 碳酸钙（CaCO3）作为贝壳的主要成分，与有机质框架相互作用形成贝壳，为贝类提供保护作用。Ca2+是CaCO3的重要组成部分，其在贝类体内的获取、转运、沉淀过程均会显著影响CaCO3沉积。然而，目前关于贝壳中碳酸钙沉积过程以及相关基因的作用机制仍不明晰。钙调蛋白（Calmodulin, CaM）是一种广泛存在于真核细胞中并与Ca2+特异性结合的蛋白，主要参与细胞信号转导、靶酶活性调控和Ca2+稳态调节等多种生理过程。为研究CaM基因与贝壳碳酸钙沉积的关系，本研究对缢蛏CaM基因（ScCaM）进行分子鉴定和表达特征分析，并研究了ScCaM重组蛋白的Ca2+结合活性及其在CaCO3沉积中的作用。结果显示，ScCaM基因共编码149个氨基酸，含有4个连续EF-hand结构域；ScCaM在各组织中均能表达，其中在鳃和外套膜组织中表达水平显著高于足、水管、闭壳肌和肝胰腺等组织（P < 0.05）；缢蛏壳碳酸钙含量与其壳重呈正比，并且贝壳重量较大的个体，其ScCaM基因表达水平较高。ScCaM重组蛋白具有钙离子结合活性，可加快碳酸钙沉淀速率，并且促进效应呈现出明显的蛋白浓度依赖性。结果表明，ScCaM基因/蛋白表达与贝壳碳酸钙含量密切相关，即ScCaM基因/蛋白表达量升高，可增强Ca2+运输效率，促进壳碳酸钙沉积，从而提高贝壳重量。本研究初步探讨了ScCaM基因在贝壳碳酸钙沉积中的作用，为解析缢蛏贝壳形成的分子机制提供了理论基础。Abstract: Calcium carbonate (CaCO3), as a major component of shells, interacts with the organic matter framework to form shells and provide protection for mollusks. Ca2+ is an important component of CaCO3, and its acquisition, transport, and precipitation processes can significantly affect calcium carbonate deposition in mollusks. However, there is still a lack of clarity regarding the process of calcium carbonate deposition and mechanism of related genes in forming shells. Calmodulin (CaM) is a protein widely found in eukaryotic cells and specifically binds to Ca2+, which is mainly involved in a variety of physiological processes such as cellular signal transduction, regulation of target enzyme activities and regulation of Ca2+ homeostasis. In order to investigate the relationship between CaM gene and calcium carbonate deposition in shells, we performed molecular identification and expression characterization of CaM gene in Sinonovacula constricta (ScCaM), and investigated the Ca2+-binding activity of the recombinant protein ScCaM and its role in calcium carbonate deposition. The results showed that the ScCaM gene encoded a total of 149 amino acids and contained four consecutive EF-hand structural domains. ScCaM was expressed in all tissues, with the expression level in gill and mantle tissues being significantly higher than in foot, siphon, adductor muscle and hepatopancreas tissues (P < 0.05). Furthermore, the content of calcium carbonate in shells was positively proportional to their shell weight. Meanwhile, individuals with larger shell weights had higher expression levels of ScCaM. ScCaM recombinant protein had calcium ion binding activity, which can accelerate the rate of calcium carbonate deposition, and the promotion effect showed an obvious protein concentration dependence. The results showed that ScCaM gene/protein expression was closely related to shell calcium carbonate content: elevated ScCaM gene/protein expression could enhance Ca2+ transport efficiency, promote shell calcium carbonate deposition, and thus increase shell weight. This study preliminarily investigated the role of ScCaM gene in shell calcium carbonate deposition, and provided a theoretical basis for analyzing the molecular mechanism of shell formation in S. constricta.
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Key words:
- Sinonovacula constricta /
- calmodulin (CaM) /
- shell weight /
- calcium carbonate /
- expression
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图 1 CaM蛋白的氨基酸多序列比对（A）、同源性分析（B）、邻接法构建系统进化树（C）和蛋白三级结构（D）
注：红色三角标记为缢蛏CaM特有氨基酸残基，红色方框标注为氨基酸多态性。蛋白三级结构各结构域以相同颜色色块标注于序列比对图中。
Fig. 1 Multiple amino acid sequence alignment of CaM proteins (A), homology analysis among CaM proteins (B), phylogenetic tree constructed by neighbour-joining method (C), and tertiary structure of CaM protein (D)
Note: Red triangle indicates specific amino acid residues in the CaM protein of S.constricta, and red box indicates amino acid polymorphism sites. The structural domains of the protein tertiary structure are annotated with the same color blocks in Fig. 1(A).
图 2 ScCaM基因在不同组织中的表达（A，n=6）、不同壳重组碳酸钙含量（B，n=30）和ScCaM基因表达（C，n=6）分析
注：不同小写字母表示差异显著 (P < 0.05)；LSW：低贝壳重量组；HSW：高贝壳重量组
Fig. 2 The relative expression level of ScCaM in various tissues (A, n=6), the calcium carbonate content (B, n=30) and ScCaM gene expression level (C, n=6) analysis in different shell weight groups.
Note: Different lowercase letters indicate significant differences (P < 0.05); LSW: low shell weight group; HSW: high shell weight group.
图 3 ScCaM基因的原核表达和蛋白纯化分析
注：A. 重组钙调蛋白原核表达： a. pET-32a空载，b. pET-32a-CaM上清，c. pET-32a-CaM沉淀，d. pET-32a-CaM全菌，M. Marker；B. 缢蛏钙调蛋白原核表达纯化： 1. pET-32a空载，2. pET-32a空载+IPTG，3. pET-32a-CaM，4. pET-32a-CaM+IPTG，5. pET-32a-CaM纯化；红框表示重组蛋白rScCaM
Fig. 3 Analysis of prokaryotic expression and protein purification of ScCaM gene
Note: A. Recombinant calmodulin prokaryotic expression results: a. pET-32a unloaded; b. pET-32a-CaM supernatant; c. pET-32a-CaM precipitation, d. pET-32a-CaM whole bacteria; M. protein marker, B. Results of purification of prokaryotic expression of S. constricta calmodulin: 1. pET-32a unloaded, 2. pET-32a unloaded + IPTG, 3. pET-32a-CaM, 4. pET-32a-CaM + IPTG, 5. pET-32a-CaM purification. Red boxes indicates recombinant protein rScCaM.
图 4 ScCaM重组蛋白的钙离子结合活性（A）及碳酸钙沉积速率分析（B）
注：A：1. rScCaM + EDTA；2. rScCaM + EDTA + MgCl2；3. rScCaM + EDTA + CaCl2；4. 空白对照rScCaM. “*”表示与PBS对照组比较差异显著 (P < 0.05)
Fig. 4 Analysis of the calcium ion binding activity of ScCaM recombinant protein (A) and its influence on the rate of calcium carbonate deposition (B)
Note: A: 1. rScCaM + EDTA; 2. rScCaM + EDTA + MgCl2; 3. rScCaM + EDTA + CaCl2; 4. blank control rScCaM. “*” indicates a significant difference compared with the PBS control group (P < 0.05).
表 1 实验所用的引物和序列
Tab. 1 Primers and sequences used in this study
引物 序列（5’-3’） 用途 CaM-F TTCTTCTGTTAGTTGATCAGCCAT CDS全长验证 CaM-R ATGGCTGATCAACTAACAGAAGAAC cCaM-F GCCATGGCTGATATCGGATCCATGGCT
GATCAACTAACAGAAGAACA克隆 cCaM-R GTGGTGGTGGTGGTGCTCGAGCTACC
GCGATGTCATCATCTTCAqRTCaM-F CGACGGTAACGGCACGATAG 荧光定量 qRTCaM-R CTTCCCGGATCATTTCATCAACC RS9-R CGTCTCAAAAGGGCATTACC RS9-F TGAAGTCTGGCGTGTCAAGT 
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