Identification of calmodulin (<i>ScCaM</i>) gene and its correlation with shell calcium carbonate deposition of <i>Sinonovacula constricta</i>

Luo Xiaoqi; Xu Hongqiang; Zhu Jie; Yao Hanhan; Dong Yinghui

Article Contents

Article Navigation > Haiyang Xuebao > 2025 > Accepted Manuscript

Luo Xiaoqi，Xu Hongqiang，Zhu Jie, et al. Identification of calmodulin (ScCaM) gene and its correlation with shell calcium carbonate deposition of Sinonovacula constricta[J]. Haiyang Xuebao，2025, 47(x)：1–10

Citation:

Luo Xiaoqi，Xu Hongqiang，Zhu Jie, et al. Identification of calmodulin (ScCaM) gene and its correlation with shell calcium carbonate deposition of Sinonovacula constricta[J]. Haiyang Xuebao，2025, 47(x)：1–10

Citation:

PDF( 8143 KB)

Identification of calmodulin (ScCaM) gene and its correlation with shell calcium carbonate deposition of Sinonovacula constricta

Luo Xiaoqi^1
,,
Xu Hongqiang^{2, 3},
Zhu Jie³,
Yao Hanhan^{1
,
,},
Dong Yinghui^{2, 3
,
,}

1.
Zhejiang Key Laboratory of Aquatic Germplasm Resources, College of Biological & Environmental Sciences, Zhejiang Wanli University, Ningbo 315100, China
2.
College of Advanced Agricultural Sciences, Zhejiang Wanli University, Ningbo 315101, China
3.
Ninghai Marine Biological Seed Industry Research Institute, Zhejiang Wanli University, Ninghai 315604, China

Received Date: 2024-12-31
Rev Recd Date: 2025-04-28

Available Online: 2025-05-15

Abstract

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.
- Sinonovacula constricta,
- calmodulin (CaM),
- shell weight,
- calcium carbonate,
- expression

FullText(HTML)

References(52)

References

[1]	Currey J D. Mechanical properties of mother of pearl in tension[J]. Proceedings of the Royal Society B: Biological Sciences, 1977, 196(1125): 443−463.
[2]	段婷婷, 郑威, 黄玉松, 等. 鲍鱼壳的跨尺度结构及性能表征[J]. 暨南大学学报(自然科学与医学版), 2018, 39(2): 105−111. Duan Tingting, Zheng Wei, Huang Yusong, et al. The multiscale structure and performance characteristics of abalone shell[J]. Journal of Jinan University (Natural Science & Medicine Edition), 2018, 39(2): 105−111.
[3]	Gilbert P U P A, Bergmann K D, Boekelheide N, et al. Biomineralization: integrating mechanism and evolutionary history[J]. Science Advances, 2022, 8(10): eabl9653. doi: 10.1126/sciadv.abl9653
[4]	Suzuki M, Murayama E, Inoue H, et al. Characterization of Prismalin-14, a novel matrix protein from the prismatic layer of the Japanese pearl oyster (Pinctada fucata)[J]. Biochemical Journal, 2004, 382(1): 205−213. doi: 10.1042/BJ20040319
[5]	Xie Jun, Liang Jian, Sun Juan, et al. Influence of the extrapallial fluid of Pinctada fucata on the crystallization of calcium carbonate and shell biomineralization[J]. Crystal Growth & Design, 2016, 16(2): 672−680.
[6]	李云娟. 池蝶蚌外套膜组织学及钙调蛋白基因的克隆和表达特性分析[D]. 南昌: 南昌大学, 2008. Li Yunjuan. Observation on the mantle, cloning and expression analysis of the calmodulin cDNA from Hyriopsis schlegeli[D]. Nanchang: Nanchang University, 2008.
[7]	Haiech J, Moulhaye S B M, Kilhoffer M C. The EF-Handome: combining comparative genomic study using FamDBtool, a new bioinformatics tool, and the network of expertise of the European Calcium Society[J]. Biochimica et Biophysica Acta (BBA)-Molecular Cell Research, 2004, 1742(1/3): 179−183.
[8]	徐友涵. 钙调蛋白的结构与功能(上)[J]. 生物化学与生物物理进展, 1985(1): 22−27. Xu Youhan. The structure and function of calmodulin (above)[J]. Progress in Biochemistry and Biophysics, 1985(1): 22−27. (查阅网上资料, 未找到对应的英文翻译, 请确认)
[9]	Stevens F C. Calmodulin: an introduction[J]. Canadian Journal of Biochemistry and Cell Biology, 1983, 61(8): 906−910. doi: 10.1139/o83-115
[10]	Zhou Xiaoying, Cui Yazhou, Luan Jing, et al. Label-free quantification proteomics reveals novel calcium binding proteins in matrix vesicles isolated from mineralizing Saos-2 cells[J]. BioScience Trends, 2013, 7(3): 144−151.
[11]	Zhang Liang, Feng Xu, McDonald J M. The role of calmodulin in the regulation of osteoclastogenesis[J]. Endocrinology, 2003, 144(10): 4536−4543. doi: 10.1210/en.2003-0147
[12]	Bhasker T V, Gowda N K S, Mondal S, et al. Boron supplementation influences bone mineralization by modulating expression of genes regulating calcium utilization[J]. Animal Nutrition and Feed Technology, 2017, 17(2): 201−215. doi: 10.5958/0974-181X.2017.00021.X
[13]	Ge Meiling, Mo Jing, Ip J C H, et al. Adaptive biomineralization in two morphotypes of Sternaspidae (Annelida) from the Northern China Seas[J]. Frontiers in Marine Science, 2022, 9: 984989. doi: 10.3389/fmars.2022.984989
[14]	Shi Yaohua, Yu Chengcheng, Gu Zhifeng, et al. Characterization of the pearl oyster (Pinctada martensii) mantle transcriptome unravels biomineralization genes[J]. Marine Biotechnology, 2013, 15(2): 175−187. doi: 10.1007/s10126-012-9476-x
[15]	Li Shuo, Xie Liping, Zhang Cen, et al. Cloning and expression of a pivotal calcium metabolism regulator: calmodulin involved in shell formation from pearl oyster (Pinctada fucata)[J]. Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology, 2004, 138(3): 235−243. doi: 10.1016/j.cbpc.2004.03.012
[16]	Peng Kou, Liu Fanglan, Wang Junhua, et al. Calmodulin highly expressed during the formation of pearl sac in freshwater pearl mussel (Hyriopsis schlegelii)[J]. Thalassas: An International Journal of Marine Sciences, 2018, 34(1): 219−225. doi: 10.1007/s41208-017-0054-x
[17]	Chen Yige, Yao Yuanbin, Shen Xiaoya, et al. Transcription profiling reveals co-regulation mechanism of gene expression related to growth and mineralization induced by pearl cultivation in Hyriopsis cumingii[J]. Frontiers in Marine Science, 2024, 11: 1443863. doi: 10.3389/fmars.2024.1443863
[18]	尚朝, 施杨, 李文娟, 等. 不同Ca²⁺浓度养殖环境下三角帆蚌外套膜和内脏团组织钙调蛋白基因的表达[J]. 生物技术通报, 2016, 32(12): 152−159. Shang Chao, Shi Yang, Li Wenjuan, et al. Expressions of calmodulin gene in Hyriopsis cumingii mantle and visceral tissues under the culture environment with different Ca²⁺ concentrations[J]. Biotechnology Bulletin, 2016, 32(12): 152−159.
[19]	Zhan Xin, Gu Zhifeng, Yu Chengcheng, et al. Expressed sequence tags 454 sequencing and biomineralization gene expression for pearl sac of the pearl oyster, Pinctada fucata martensii[J]. Aquaculture Research, 2015, 46(3): 745−758. doi: 10.1111/are.12227
[20]	Richards M, Xu Wei, Mallozzi A, et al. Production of calcium-binding proteins in Crassostrea virginica in response to increased environmental CO₂ concentration[J]. Frontiers in Marine Science, 2018, 5: 203. doi: 10.3389/fmars.2018.00203
[21]	Teixidó N, Caroselli E, Alliouane S, et al. Ocean acidification causes variable trait-shifts in a coral species[J]. Global Change Biology, 2020, 26(12): 6813−6830. doi: 10.1111/gcb.15372
[22]	Sleight V A, Thorne M A S, Peck L S, et al. Transcriptomic response to shell damage in the Antarctic clam, Laternula elliptica: time scales and spatial localisation[J]. Marine Genomics, 2015, 20: 45−55. doi: 10.1016/j.margen.2015.01.009
[23]	Nogueira D J, Mattos J J, Dybas P R, et al. Effects of phenanthrene on early development of the Pacific oyster Crassostrea gigas (Thunberg, 1789)[J]. Aquatic Toxicology, 2017, 191: 50−61. doi: 10.1016/j.aquatox.2017.07.022
[24]	Faas G C, Raghavachari S, Lisman J E, et al. Calmodulin as a direct detector of Ca²⁺ signals[J]. Nature Neuroscience, 2011, 14(3): 301−304. doi: 10.1038/nn.2746
[25]	农业农村部渔业渔政管理局, 全国水产技术推广总站, 中国水产学会. 2024中国渔业统计年鉴[M]. 北京: 中国农业出版社, 2024. Ministry of Agriculture and Rural Affairs of the People’s Republic of China, National Fisheries Technology Extension Center, China Society of Fisheries. China Fishery Statistical Yearbook[M]. Beijing: China Agriculture Press, 2024.
[26]	Lombardi S A, Chon G D, Lee J J W, et al. Shell hardness and compressive strength of the eastern oyster, Crassostrea virginica, and the Asian oyster, Crassostrea ariakensis[J]. The Biological Bulletin, 2013, 225(3): 175−183. doi: 10.1086/BBLv225n3p175
[27]	Hamilton D J, Nudds T D, Neate J. Size-selective predation of blue mussels (Mytilus edulis) by common eiders (Somateria mollissima) under controlled field conditions[J]. The Auk, 1999, 116(2): 403−416. doi: 10.2307/4089374
[28]	Parveen S, Chakraborty A, Chanda D K, et al. Microstructure analysis and chemical and mechanical characterization of the shells of three freshwater snails[J]. ACS Omega, 2020, 5(40): 25757−25771. doi: 10.1021/acsomega.0c03064
[29]	莫天宝, 徐洪强, 何京, 等. 五种双壳贝类贝壳微观结构观察与成分分析[J]. 海洋科学, 2022, 46(12): 41−49. Mo Tianbao, Xu Hongqiang, He Jing, et al. Microstructure and composition analysis of five species of economic bivalves[J]. Marine Sciences, 2022, 46(12): 41−49.
[30]	Waterhouse A M, Procter J B, Martin D M A, et al. Jalview Version 2-a multiple sequence alignment editor and analysis workbench[J]. Bioinformatics, 2009, 25(9): 1189−1191. doi: 10.1093/bioinformatics/btp033
[31]	Clewley J P, Arnold C. MEGALIGN: the multiple alignment module of LASERGENE[M]//Swindell S R. Sequence Data Analysis Guidebook. Totowa: Springer, 1997: 119−129.
[32]	徐海龙, 王芮, 许莉莉, 等. 6种海洋双壳类贝壳中碳酸钙、碳酸镁含量的测定[J]. 海洋通报, 2016, 35(4): 421−426. doi: 10.11840/j.issn.1001-6392.2016.04.009 Xu Hailong, Wang Rui, Xu Lili, et al. Determination of calcium carbonate and magnesium carbonate in the shell of six marine bivalves[J]. Marine Science Bulletin, 2016, 35(4): 421−426. doi: 10.11840/j.issn.1001-6392.2016.04.009
[33]	周景道, 钱智光, 田芳, 等. 建议使用紫脲酸铵——萘酚绿B新混合指示剂[J]. 淮北煤师院学报, 1995, 16(3): 25−27. Zhou Jingdao, Qian Zhiguang, Tian Fang, et al. Naphthalene phenol green as new nixed indicator[J]. Journal of Huaibei Coal Mining Teachers College, 1995, 16(3): 25−27.
[34]	Livak K J, Schmittgen T D. Analysis of relative gene expression data using real-time quantitative PCR and the $ 2^{-\Delta \Delta C_{\mathrm{T}}} $ method[J]. Methods, 2001, 25(4): 402−408. doi: 10.1006/meth.2001.1262
[35]	辛晓雨. 海洋酸化下长牡蛎外套膜对钙质壳形成的调控及机制的初探[D]. 大连: 大连海洋大学, 2023. Xin Xiaoyu. Preliminary study on the regulation of calcareous shell formation by mantle under ocean acidification and its mechanism in oyster Crassostrea gigas[J]. Dalian: Dalian Ocean University, 2023.
[36]	熊新威. 马氏珠母贝珍珠层基质蛋白的相互作用模式及其功能研究[D]. 湛江: 广东海洋大学, 2022. Xiong Xinwei. Interaction patterns of nacre matrix proteins and its functional studies in Pinctada fucata martensii[D]. Zhanjiang: Guangdong Ocean University, 2022.
[37]	张明霞, 张科, 袁凤娟, 等. 背角无齿蚌钙调蛋白基因的克隆及Ca²⁺和Cd²⁺对其表达的影响[J]. 生态毒理学报, 2021, 16(3): 227−238. doi: 10.7524/AJE.1673-5897.20200423001 Zhang Mingxia, Zhang Ke, Yuan Fengjuan, et al. Characterization of AwCaM1 from freshwater clam Anodonta woodiana and effect of Ca²⁺ and Cd²⁺ on its expressions[J]. Asian Journal of Ecotoxicology, 2021, 16(3): 227−238. doi: 10.7524/AJE.1673-5897.20200423001
[38]	李素萍, 喻达辉, 李应清, 等. 香港牡蛎钙调蛋白基因的克隆和组织表达分析[J]. 安徽农业科学, 2022, 50(24): 75−81. doi: 10.3969/j.issn.0517-6611.2022.24.019 Li Suping, Yu Dahui, Li Yingqing, et al. Molecular cloning and expression analysis of Calmodulin gene in Crassostrea hongkongensis[J]. Journal of Anhui Agricultural Sciences, 2022, 50(24): 75−81. doi: 10.3969/j.issn.0517-6611.2022.24.019
[39]	Ivanina A V, Borah B, Rimkevicius T, et al. The role of the vascular endothelial growth factor (VEGF) signaling in biomineralization of the oyster Crassostrea gigas[J]. Frontiers in Marine Science, 2018, 5: 309. doi: 10.3389/fmars.2018.00309
[40]	Li Shuo, Xie Liping, Ma Zhuojun, et al. cDNA cloning and characterization of a novel calmodulin-like protein from pearl oyster Pinctada fucata[J]. The FEBS Journal, 2005, 272(19): 4899−4910. doi: 10.1111/j.1742-4658.2005.04899.x
[41]	Stommel E W, Stephens R E, Masure H R, et al. Specific localization of scallop gill epithelial calmodulin in cilia[J]. The Journal of Cell Biology, 1982, 92(3): 622−628. doi: 10.1083/jcb.92.3.622
[42]	Brini M, Carafoli E, Calì T. The plasma membrane calcium pumps: focus on the role in (neuro)pathology[J]. Biochemical and Biophysical Research Communications, 2017, 483(4): 1116−1124. doi: 10.1016/j.bbrc.2016.07.117
[43]	Zeng L G, Wang J H, Li Y J, et al. Molecular characteristics and expression of calmodulin cDNA from the freshwater pearl mussel, Hyriopsis schlegelii[J]. Genetics and Molecular Research, 2012, 11(1): 42−52. doi: 10.4238/2012.January.9.5
[44]	赵鲁苹, 徐焕志, 陈东, 等. 厚壳贻贝贝壳的微结构及光谱分析[J]. 浙江大学学报(理学版), 2015, 42(3): 339−346. Zhao Luping, Xu Huanzhi, Chen Dong, et al. Microstructure and spectral analysis of Mytilus coruscus shell[J]. Journal of Zhejiang University (Science Edition), 2015, 42(3): 339−346.
[45]	Wheeler A P, George J W, Evans C A. Control of calcium carbonate nucleation and crystal growth by soluble matrx of oyster shell[J]. Science, 1981, 212(4501): 1397−1398. doi: 10.1126/science.212.4501.1397
[46]	李兴霞. 长牡蛎左右外套膜的分子差异研究[D]. 沈阳: 沈阳农业大学, 2017. Li Xingxia. Study on the molecular difference of the mantle of pacific oyster Crassostrea gigas[D]. Shenyang: Shenyang Agricultural University, 2017.
[47]	Yan Zhenguang, Jing Gu, Gong Ningping, et al. N40, a novel nonacidic matrix protein from pearl oyster nacre, facilitates nucleation of aragonite in vitro[J]. Biomacromolecules, 2007, 8(11): 3597−3601. doi: 10.1021/bm0701494
[48]	Fang Dong, Pan Cong, Lin Huijuan, et al. Novel basic protein, PfN23, functions as key macromolecule during nacre formation[J]. Journal of Biological Chemistry, 2012, 287(19): 15776−15785. doi: 10.1074/jbc.M112.341594
[49]	孔晶晶. 基质蛋白调控晶体自组装形成贝壳有序结构的机制研究[D]. 北京: 清华大学, 2021. Kong Jingjing. Studies on the mechanism of matrix proteins regulating crystals self-assembly to form ordered shell structure[D]. Beijing: Tsinghua University, 2021.
[50]	Fang Zi, Yan Zhenguang, Li Shuo, et al. Localization of calmodulin and calmodulin-like protein and their functions in biomineralization in P. fucata[J]. Progress in Natural Science, 2008, 18(4): 405−412. doi: 10.1016/j.pnsc.2007.11.011
[51]	于旭蓉, 仇雪梅, 常亚青, 等. 长牡蛎钙调蛋白基因克隆及多态性分析[J]. 南方农业学报, 2012, 43(6): 855−860. doi: 10.3969/j:issn.2095-1191.2012.06.855 Yu Xurong, Qiu Xuemei, Chang Yaqing, et al. Cloning and polymorphism analysis of calmodulin (CaM) gene obtained from Crassostrea gigas[J]. Journal of Southern Agriculture, 2012, 43(6): 855−860. doi: 10.3969/j:issn.2095-1191.2012.06.855
[52]	Xin Xiaoyu, Liu Chang, Liu Zhaoqu, et al. Calmodulin regulates the calcium homeostasis in mantle of Crassostrea gigas under ocean acidification[J]. Frontiers in Marine Science, 2022, 9: 1050022. doi: 10.3389/fmars.2022.1050022