半滑舌鳎(Cynoglossus Semilaevis)EB1基因的克隆、表达及其对细胞迁移能力的相关性分析

Cloning,Expression Analysis and Cell Migration Analysis of EB1 in Cynoglossus Semilaevis

EB1作为一种微管末端结合蛋白,在微管聚合和微管正端蛋白复合体的装配中起着关键的作用。以半滑舌鳎cDNA文库为基础,克隆EB1基因。该基因包含777bp的开放读码框,编码推测的氨基酸258个,分子量为29.497kDa。与其他物种EB1序列同源比对发现,该蛋白包括EB1和CH 2个保守的结构域,以及FYF和EEF/Y 2个保守的三联氨基酸位点。同源聚类分析结果显示,EB1蛋白的系统进化较好地反映物种从低等到高等的进化进程。实时荧光定量PCR结果表明,该基因在脑、鳃、心脏、肠、头肾、肝脏、肌肉、卵巢、脾脏和精巢组织中都有表达,其中,卵巢的表达量最高,比表达量最低的肝组织高14.6倍。对细胞迁移能力的研究中发现,细胞迁出能力显示出与荧光定量PCR中选取的组织中EB1的表达量相符的规律,EB1基因表达量越高的组织细胞迁出的能力越强。

EB1, as a microtubule plus end binding protein, plays an important role in the microtubule polymerization and microtubules equipment found on the protein complex. In current experiment, EB1 gene was cloned on the basis of half smooth tongue sole cDNA library. The gene contained an open reading frame of 777 bp code that encoded putative 258 amino acids with a calculated molecular mass of 29. 497 kDa. The deduced amino acid sequence was aligned with other species, and they had the high identity and homology. It was found that the proteins included two conservative domain structure, CH and EB1, and two conservative trigeminy amino acid sites, FYF and EEF/Y. Homologous clustering analysis results showed that the EB1 proteins could well reflect the species development of evolution from lower to higher states. Real-time fluorescent quantitative PCR results showed that the genes expressed in all the tissues selected and, the highest level existed in ovary, which was higher 14.6 times than the lowest amount of liver tissue. Consistent with qRT-PCR results, cell mobility showed consistency with the expression of EB1 in tissues.