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作 者 ：吴卫君, 刘士力, 李倩, 王雨辰, 练青平, 胡廷尖, 贾永义, 蒋文枰, 李喜莲

期 刊 ：生物技术通报 2016年 32卷 5期 页码：114-123

关键词：角蛋白18(K18)；cDNA克隆；泥鳅(Misgurnus anguillicaudatus)；定量表达；

Keywords:keratin 18（K18）, cDNA clone, loach（Misgurnus anguillicaudatus）, quantitative expression,

摘 要 ：角蛋白18（K18）是角蛋白的一种，为中间纤维蛋白，是细胞骨架的组成部分。为了解泥鳅（Misgurnus anguillicaudatus）K18序列特征及其在不同组织中的表达，研究采用3'-和5'-RACE法克隆K18基因cDNA全长序列，并运用实时荧光定量PCR技术探讨其在不同组织中的表达。结果表明，泥鳅K18基因cDNA全长序列1 694 bp，其中包含开放阅读框1 299 bp，编码432个氨基酸，其序列具有Ⅰ型角蛋白序列DGKVVS，以及非常相似序列DNA（R/K）LAADDF。相似度分析显示，泥鳅K18与大鳞副泥鳅（Paramisgurnus dabryanus）的相似度最高为98%，与其它物种K18也存在不同程度的相似度。系统进化分析表明，泥鳅与其他物种的K18系统发育同源，且同陆生脊椎动物和硬骨鱼类同源，并与大鳞副泥鳅的关系最为密切。通过定量分析，K18基因在泥鳅肠、肌肉、心脏、胃、肝脏、精巢、卵巢、脾脏等8种组织都有表达，其中，肠表达最高，心脏最低。统计学分析表明，K18基因的表达在不同组织间存在显著性差异。

Abstract:Keratin 18（K18），one of the keratin，is the intermediate filament proteins and an integral part of the cytoskeleton. In order to understand the sequence characteristics and expression of K18，cDNA full-length sequence of K18 gene was cloned from loach（Misgurnus anguillicaudatus）using RACE，and its expression in different tissues was explored using real-time quantitative PCR. The results showed that the cDNA full-length of cloned K18 gene was 1 694 bp，containing a 1 299 bp open reading frame encoding 432 amino acids. There were the sequence DGKVVS，and the very similar sequence DNA（R/K）LAADDF of type I keratin. Identity analysis revealed that loach had the highest identity up to 98% with large-scale loach（Paramisgurnus dabryanus）in K18，and there were various levels of identity with other species. Phylogenetic analysis demonstrated that the K18 gene of loach was in phylogenetic homology with other species，and in true orthologs with terrestrial vertebrates（tetrapods）and teleosts，moreover，closest with large-scale loach. K18 gene was expressed in the 8 tissues of intestine，muscle，heart，stomach，liver，testis，ovary，spleen，the highest in the intestine，and the lowest in the heart. Statistical analysis showed that the differences of expression among different tissues were significant.

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