大菱鲆T淋巴细胞酪氨酸激酶(LCK)基因全长cDNA的克隆及表达分析  

Cloning,characterization and expression analysis of a LCK gene from turbot (Scophthalmus maximus)

作　　者：王贤丽[1,2] 张玉喜[2] 孟亮[2] 陈松林[2] 

机构地区：[1]中国海洋大学海洋生命学院,山东青岛266003 [2]农业部海洋渔业资源可持续利用重点开放实验室,中国水产科学研究院黄海水产研究所,山东青岛266071

出　　处：《中国水产科学》2009年第5期660-667,共8页Journal of Fishery Sciences of China

基　　金：国家973计划项目(2004B117403);国家自然科学基金项目(40376047)

摘　　要：本研究采用cDNA末端快速扩增技术(RACE)从大菱鲆(Scophthalmus maximus)脾脏cDNA文库中克隆得到T淋巴细胞酪氨酸激酶(LCK)全长cDNA序列。该序列包含193bp的5′末端非编码区(5′UTR),1506bp的开放阅读框(ORF)和300bp3′UTR,整个开放阅读框编码502个氨基酸。系统发生分析表明,大菱鲆LCK基因与红鳍东方鲀(Fugu rubripes)和黑青斑河鲀(Tetraodon nigroviridis)的亲缘关系最近。在大菱鲆正常组织、胚胎细胞(TEC)和鳗弧菌(Vibrio anguillarum)感染的免疫器官组织中对LCK基因进行了RT-PCR表达分析。结果表明,大菱鲆LCK基因只在正常脾脏组织中表达;在用鳗弧菌感染12h后,大菱鲆胚胎细胞LCK基因表达增强;在鳗弧菌感染的大菱鲆免疫组织中,只在脾脏中检测到LCK基因表达,鳗弧菌感染48h后,LCK基因在脾脏中表达最强。这些结果表明,LCK基因在大菱鲆脾脏免疫应答中起着重要作用。The lymphocyte cell kinase（ LCK） is a member of the Src family kinases of protein tryrosine kinase. Turbot （Scophthalmus maximus） is one of the most commercially important marine fish in China. To detect whether LCK gene is specifically expressed in the turbot immunity organs is very important for further investigation on the functions of LCK in turbot immunity response. In the present research,a novel turbot LCK gene was cloned from a turbot spleen cDNA library using RACE method. After splicing and assembling analysis with DNASTAR SeqMan software, a full-length LCK cDNA fragment of 1 999 bp was obtained, which included 193 bp 5＇ terminal untranslated region （UTR）, 1 506 bp encoding region and 300 bp 3＇ terminal UTR containing one typical tailing signal （AATAAA） followed by the poly （A） tail. Amino acid sequence of turbot LCK was deduced from the nucleotide sequence of LCK cDNA. The open reading frame （ORF） of 1 506 bp was found to code for a protein of 502 amino acid residues. Phylogenetic analysis showed that the deduced LCK clustered with fugu rubripes and spotted green pufferfish （Tetraodon nigroviridis）. RT-PCR was conducted to detect the expression of LCK gene in various tissues of healthy turbots, the infected turbot embryonic cells （TEC） and immunity tissues of turbot infected with Vibrio anguillarum. RT-PCR result showed that turbot LCK gene was only expressed in spleen of uninfected turbot and no expression was found in intestine, gill, muscle, kidney, gonad, heart, brain and skin. In TECs the expression of LCK dramatically increased after 12 h of challenge with V. anguillarum. The expression of the turbot LCK cDNA was analyzed in liver, spleen and head kidney after challenge with V. anguillarum. In liver and head kidney, no expression of LCK was found. Furthermore, the turbot LCK was highly induced in spleen after 48 h of challenge with V. anguillarum. These results indicate that LCK plays an important role in turbot immune response.

关 键 词：大菱鲆(Scophthal musmaximus) T淋巴细胞酪氨酸激酶 CDNA RT-PCR 

分 类 号：Q959[生物学—动物学]