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瘤背石磺肌球蛋白重链(MyHC)基因的克隆与表达分析
顾冰宁,刘欣,沈和定,王冬凤,杨铁柱,朱敏,史艳梅,李柏航
上海海洋大学水产种质资源发掘与利用教育部重点实验室 水产科学国家级实验教学示范中心 海洋动物系统分类与进化上海高校重点实验室 上海 201306
摘要:
肌球蛋白是动物体内重要的功能性马达蛋白,调控机体的信号传导、肌肉收缩和细胞器运动,为探究肌肉在瘤背石磺(Onchidium struma)由海洋向陆地进化过程中发挥作用的分子机制,实验以石磺科4种贝类转录组数据为基础,采用RACE方法从瘤背石磺肌肉中首次克隆到肌球蛋白重链(Myosin heavy chain, MyHC)基因cDNA的全长并进行组织表达分析。研究结果显示,瘤背石磺MyHC基因cDNA全长为7566 bp,包括5895 bp的开放阅读框,228 bp的5端非翻译区,1443 bp的3端非翻译区,共编码1964个氨基酸。预测该基因编码的蛋白质由31713个原子组成,分子式为C9765H15897N2849O3150S52,分子量约为225.28 kDa,理论等电点为5.56,N端信号肽具有29个氨基酸长度。瘤背石磺MyHC具有2个保守结构域MYSc_class_Ⅱ和Myosin_tail_l,且亲水性氨基酸集中在Myosin_tail_l区。系统进化树分析显示,瘤背石磺MyHC与光滑双脐螺(Biomphalaria glabrata) MyHC的亲缘关系最近。RT-PCR结果显示,MyHC基因在各个组织中均有表达,腹足和背部皮肤表达量最高,腹部皮肤、口球、肺囊中高表达,肝胰腺、蛋白腺、两性腺中微量表达(P<0.05)。MyHC基因在瘤背石磺的主要运动器官腹足中高表达,说明肌肉运动对瘤背石磺湿地环境的两栖适应性具有至关重要的作用。实验结果为今后进一步研究瘤背石磺肌球蛋白重链基因进行原核表达及多克隆抗体的制备奠定了良好基础,更为深入探讨海洋无脊椎动物从海洋向陆地进化的研究提供有参考意义的分子依据。
关键词:  瘤背石磺  MyHC  基因克隆  组织表达
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Molecular cloning and analysis of the MyHC gene in Onchidium struma
GU Bingning1,2,3, LIU Xin1,2,3, SHEN Heding1,2,3, WANG Dongfeng1,2,3, YANG Tiezhu1,2,3, ZHU Min1,2,3, SHI Yanmei1,2,3, LI Bohang1,2,3
1.Key Laboratory of Exploration and Utilization of Aquatic Genetic Resources, Ministry of Education;2.National Demonstration Center for Experimental Fisheries Science Education;3.Shanghai Universities Key Laboratory of Marine Animal Taxonomy and Evolution, Shanghai Ocean University, Shanghai 201306
Abstract:
Onchidium struma belongs to Mollusca, Pulmonata, and was regarded as the transitional taxa from water to land because of its amphibious characteristics. Having similar characters to amphibians, O. struma is a good subject for the study of marine invertebrates extending landward. As the basic component of muscle units, myosin directly influences the muscle growth and meat quality via its molecular diversity and composition. It is a ubiquitous eukaryotic motor protein that interacts with actin to generate the force for cellular movements, ranging from cytokinesis to muscle contraction. In this study, we used rapid amplification cDNA ends (RACE) methods to obtain the full-length cDNA of the myosin heavy chain (MyHC) gene in O. struma. We performed bioinformatic and expression pattern analysis of MyHC mRNA in different tissues detected by real-time fluorescent quantitative PCR (qRT-PCR). The full length of the MyHC cDNA sequence consists of 7566 base pairs (bp) comprising a 228 bp 5' untranslated region (UTR), a 1443 bp 3' UTR, and a 5895 bp open reading frame (ORF) which encodes 1964 amino acids. MyHC gene was expressed in various tissues; its highest expression was found in the foot and its lowest in the hepatopancreas (P<0.05). Moreover, the MSTN protein was predicted to be composed of 31,713 atoms and its formula is C9765H15897N2849O3150S52, with a calculated relative molecular weight of 225.28 kDa and a pI of 5.56. The result of signal peptide prediction shows that the N-terminal has a signal peptide of 29 amino acids in length. Additionally, common features were found in the MyHC of O. struma, including MYSc class II and myosin tail l domain. Molecular phylogenetic analysis shows that O. struma is closely related to Biomphalaria glabrata. This study provided a novel myosin heavy chain gene sequence in O. struma and the results indicate that the MyHC gene is important for the growth and development of this animal, as well as its muscle characterization. Furthermore, the results revealed that MyHC is not only an essential structural protein, but also a functional protein in O. struma. Conclusively, MyHC is an excellent candidate gene for studying biological evolution.
Key words:  Onchidium struma  MyHC  Gene cloning  Tissue expression