【摘要】 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。更多还原

【Abstract】 Hyriopsis cumingii is an important pearl-breeding mussel in China, and it grows fast and breeds pearls of good quality. In this study, We cloned a full-length cDNA sequence of myosin essential light chain gene(MELC) which was isolated from the mantle of Hyriopsis cumingii according to homology cloning strategy and SMART RACEPCR technique. The full length cDNA sequence was 1 119 bp, comprising a complete open reading frame of 468 bp which encoded 155 aa of 17.49 kD and pI at 4.53. The GenBank accession number was JX275828. MELC included two consertive EF-hand Ca2+-binding domains which analysed by PROSITE tool, and which belonged to EF-hand Ca2+-binding protein family. The amino acid sequences of MELC gene possessed 69%, 69% and 68% identity with Pinctada fucata, Crassostrea gigas and Haliotis discus discus, respectively. Quantitative Real-time PCR analyses showed that MELC gene was ubiquitously expressed in all tissues of H. cumingi, including adductor muscle, mantle, gill, axe foot, gonad, heart, liver, gut, especially strongly expressed in tissues related with calcium metabolism, such as mantle, gill and axe foot, which were higher than liver and gonad. The result implied MELC gene might take an important part in the calcium metabolic process of H. cumingii. Moreover, the expression of MELC gene in mantle of H. cumingii went up and came down along with the rising of Ca2+ concentration, then reached a climax at 60 mg/L, which demonstrated that the suitable Ca2+ concentration would accelerated the high expression of MELC gene, otherwise, it might inhibit the expression of MELC gene. All these results indicate MELC gene in H. cumingii may play an important role in Ca2+ uptake, transport, storage, and participate in the formation of pearls.更多还原