鲤鱼硒蛋白W基因的克隆、序列分析及其组织表达  

Molecular Cloning,Sequence Analysis and Tissue Expression of Selenoprotein W in Common Carp

作　　者：陈文波[1] 董海燕[2] 张珍[1] 蒋晓雪[1] 

机构地区：[1]河南理工大学资源环境学院,河南焦作454000 [2]湖州师范学院医学院微生物与免疫学研究所,浙江湖州313000

出　　处：《四川动物》2014年第6期893-900,共8页Sichuan Journal of Zoology

基　　金：国家自然科学基金项目(31101878);河南理工大学杰出青年科学基金项目(J2014-02);河南理工大学青年骨干教师项目(649263);河南理工大学博士基金项目(B2010-7)

摘　　要：硒蛋白是一类含有硒代半胱氨酸（Selenocysteine,Sec）的蛋白质,具有抗氧化和免疫调节等多种生物学功能。尽管目前已知硒对鱼类生长发育、抗氧化和免疫等方面有着重要的作用,但是有关鱼类硒蛋白的研究还不全面,特别是对鱼类硒蛋白W（Selenoprotein W,Sel W）的结构和功能还知之甚少。为了深入研究鱼类硒蛋白的结构特征和生理作用,本文利用RT-PCR和RACE技术从肝胰脏中克隆获得了鲤鱼Cyprinus carpio Sel W的c DNA全长序列（cc Sel W）。cc Sel W c DNA全长704 bp,含有261 bp的开放阅读框,编码86个氨基酸。预测的cc Sel W分子量为9.5 k D,在其N-端含有特征序列C-X-X-U基序,且第13位氨基酸为13Sec。在cc Sel W c DNA 3’-UTR区域含有一个89 bp的Sec插入序列（Sec-insertion sequence,SECIS）,形成经典的AUGA-AA-GA型SECIS。由于在其SECIS二级结构的顶环上有一个额外的小茎-环结构,所以鲤鱼SECIS属于Ⅱ型SECIS结构。cc Sel W与斑马鱼Danio rerio和热带爪蟾Xenopus tropicalis的同源性最高,均为84.7%,与其他物种的同源性在54%~60%之间。系统发育树结果显示,cc Sel W和斑马鱼、两栖类位于同一分支上。RT-PCR结果显示,cc Sel W mRNA在所检测的11个组织中均有表达,其中以精巢中的表达量为最高,下丘脑和肠中表达量次之,其余组织中表达量大致相当,低于精巢、下丘脑和肠。这些结果丰富了鱼类Sel W的研究内容,为进一步研究其结构和功能奠定了基础。Selenoprotein was a kind of protein that contained selenocysteine which played an important role in animals.Although selenium（ Se） was very important for fish’s growth,development,antioxidant and immunity,there were few studies about selenoproteins in fish,especially on the structure and function of selenoprotein（ Sel W）. The full-length of Sel W c DNA（ cc Sel W） was studied in common carp（ Cyprinus carpio） by cloning from hepatopancreas through RT-PCR and RACE techniques. The results indicated that the cc Sel W c DNA sequence was 704 bp,containing a 5’-untranslated region（ UTR） of 47 bp,a 3’-UTR of 396 bp with a poly（ A） tail,and an open reading frame of 261 bp encoding a polypeptide of86 amino acids. And the cc Sel W had several conserved features including the C-X-X-U motif and selenocysteine（ Sec） in site 13. The tertiary structure model of cc Sel W was constructed by SWISS-MODEL based on the template of mouse Sel W. Aβ-α-β-β-β-α secondary structure pattern,which was similar to mouse,was also displayed. An 89 bp SECIS element,which formed a classic AUGA-AA-GA secondary structure,was identified in the 3’-UTR of cc Sel W c DNA by using the program SECISearch 2. 19. Because of the additional stem-loop structure on the top of the apical loop,the SECIS of common carp should be classified as type Ⅱ. Common carp had the highest homology to Danio rerio and Xenopus tropicalis（ 84. 7%）,but relatively lower to other species（ 54% ~ 60%）. The phylogenetic tree which was constructed by neighborjoining method with 1000 replicates indicated that,cc Sel W was clustered into the same branch with D. rerio and X. tropicalis. The result of semi-quantitative RT-PCR revealed that cc Sel W mRNA was expressed in all detected tissues. The highest expression level was observed in testis,followed by hypothalamus and intestine. Our research enriched the knowledge of Sel W in fish,and provided a foundation for illuminating its structure and physiological function.

关 键 词：鲤鱼 硒蛋白W 克隆 组织表达 

分 类 号：S917.4[农业科学—水产科学]