瘤背石磺Os-NMHC的克隆、表达分析以及NMHC进化节点探讨  

Discussion on the Cloning,Expression Analysis and NMHC Evolutionary Node of Os-NMHC from Onchidium struma

作　　者：李杰[1] 史艳梅[1] 沈和定[1] 杨铁柱[1] 顾冰宁 许国绿 Li Jie;Shi Yanmei;Shen Heding;Yang Tiezhu;Gu Bingning;Xu Guolv(Key Laboratory of Exploration and Utilization of Aquatic Genetic Resources Ministry of Education, Shanghai Ocean University, Shanghai, 201306)

机构地区：[1]上海海洋大学省部共建水产种质资源发掘与利用教育部重点实验室

出　　处：《基因组学与应用生物学》2018年第5期1887-1899,共13页Genomics and Applied Biology

基　　金：国家自然科学基金项目(No.41276157)资助

摘　　要：本研究以石磺科贝类转录组数据库为基础,克隆出一条Ⅱ型非肌肉型肌球蛋白重链（NMHCⅡ）基因的全长c DNA,将其命名为Os-NMHC。该序列全长为6 057 bp,包括5 733 bp的开放阅读框,236 bp的5＇端非翻译区,88 bp的3＇端非翻译区,共编码1 910个氨基酸。预测蛋白质等电点为5.26,相对分子质量为220.46 k D。通过Phyre2程序和Pymol软件构建蛋白质三级结构图,预测蛋白质二级结构以α螺旋和β折叠为主;通过Clustalx和dna MAN软件进行氨基酸序列比对,发现其与爪蟾NMHCⅡA序列一致度为60.37%,其与爪蟾NMHCⅡB序列相似度为61.47%;通过Bayes软件构建系统进化树,发现其与加州海兔亲缘关系最近。通过q RT-PCR技术检测,发现该基因在四种石磺的背部、腹部、腹足部、肺囊、心脏、神经节中均有不同程度的表达。种间表达水平与进化趋势相一致,依次为：瘤背石磺≥平疣桑椹石磺〉里氏拟石磺〉紫色疣石磺。In this research,a full-length c DNA of nonmuscle myosin heavy chain Ⅱ（NMHC Ⅱ） named as Os-NMHC was cloned based on the transcriptome database of Onchidiidae.The length of this sequence was 6 057 bp,including a 5 733 bp open reading frame,a 236 bp 5＇-noncoding region and an 88 bp 3＇-noncoding region,encoding a total of 1 910 amino acids,.It was predicted that the protein isoelectric point was 5.26,the molecular weight was 220.46 k D.The tertiary structure of protein was constructed by Phyre2 program and Pymol software,and it was predicted that the secondary structure of protein was mainly composed of α-helix and β-sheet.The comparison of amino acid sequences was conducted by Clustalx and dna MAN software,and it was found that Os-NMHC had similarities with NMHC Ⅱ A and NMHC Ⅱ B of Xenopus leavisis at 60.37% and 61.47%respectively.The phylogenetic tree constructed by Bayes software showed that Onchidium struma had the closest relationship with Aplysia California.The results of QRT-PCR detection indicated that the expression of Os-NMHC was different in the back,abdomen,pleopod,lung-sac,heart and ganglion of four kinds of Onchidiids.The level of interspecific expression was consistent with the evolutionary trend,and the descending order was as follows,Onchidium struma≥Platevindex mortoniParaoncidium reevesii〉Peronia verruculata.

关 键 词：Os-NMHC 石磺 NMHC亚型进化 QRT-PCR 

分 类 号：S917.4[农业科学—水产科学]