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肌球蛋白必需轻链基因是EF-hand钙结合蛋白家族的成员,具有很强的Ca2+结合活性。为了研究三角帆蚌钙代谢基因调控机制,本研究采用同源克隆策略和RACE技术,从三角帆蚌外套膜组织中成功克隆得到肌球蛋白必需轻链基因(myosin essential light chain,MELC)的全长cDNA序列,共1119bp,开放阅读框468bp(47~514),编码155个氨基酸,相对分子量为17.49kD,理论等电点为4.53,基因序列提交GenBank的登录号为JX275828。通过PROSITEtools软件预测显示三角帆蚌MELC基因有一对保守的EF-hand结构域,可结合Ca2+,属于EF-hand钙结合蛋白家族。同源性比对分析显示,三角帆蚌MELC基因与合浦珠母贝(Pinctada fucata)、太平洋牡蛎(Crassostrea gigas)、盘鲍(Haliotis discus discus)等海水贝类的MELC基因同源性最高,依次为69%、69%和68%。经实时荧光定量PCR检测,发现MELC基因在闭壳肌、外套膜、鳃、斧足、性腺、心脏、肝脏、肠等8个组织中均有表达,其中在外套膜、鳃和斧足等与贝类钙代谢相关的组织中表达量远高于肝脏、肠等消化器官,预示MELC基因可能参与三角帆蚌的钙代谢过程。不同Ca2+浓度处理实验的结果表明,随着水体中Ca2+浓度逐渐升高,三角帆蚌MELC基因在外套膜中的表达量呈先上调后下降的趋势,在60mg/L时达到最高值,表明适宜的Ca2+浓度可以刺激MELC基因大量表达,而过高或过低的Ca2+浓度均会抑制MELC基因表达。实验结果表明三角帆蚌MELC基因与Ca2+吸收、转运与贮存相关,可能参与珍珠等生物矿化过程。
The myosin essential light chain gene is a member of the EF-hand calcium binding protein family and has strong Ca2 + binding activity. In order to study the regulatory mechanism of calcium metabolic genes in Hyriopsis cumingii, we used the homologous cloning strategy and RACE technique to clone myosin essential light chain (MELC) successfully from the mantle tissue of Hyriopsis cumingii A total of 1119bp long open reading frame (ORF) 468bp (47-514) encoding a polypeptide of 155 amino acids with a molecular weight of 17.49 kD and a theoretical isoelectric point of 4.53 was obtained. The sequence of the cDNA was submitted to GenBank with the accession number of JX275828. Predicted by PROSITEtools software, there is a pair of conserved EF-hand domains in the hyacinth MELC gene that bind to Ca2 + and belong to the EF-hand calcium-binding protein family. Homology comparison showed that MELC gene of Hyriopsis cumingii had the highest homology with MELC gene of sea shellfish such as Pinctada fucata, Crassostrea gigas and Haliotis discus discus, 69%, 69% and 68%. The results of real-time fluorescent quantitative PCR showed that the MELC gene was expressed in 8 tissues, including adductor muscle, mantle, gill, axillary, gonad, heart, liver and intestine, The expression level of calcium metabolism in shellfish was much higher than that in the liver, intestine and other organs, indicating that MELC may be involved in the calcium metabolism of Hyriopsis cumingii. The results of different concentrations of Ca2 + treatment showed that with the increase of Ca2 + concentration in water, the expression level of MELC gene in the mantle decreased firstly and then decreased, reaching the highest value at 60mg / L, indicating that it is suitable Ca2 + concentration can stimulate the expression of MELC gene, while over and under Ca2 + concentration can inhibit MELC gene expression. The experimental results showed that the MELC gene of Hyriopsis cumingii was related to Ca2 + absorption, transport and storage, which may be involved in the bio-mineralization process of pearl.