中国鲎β-actin基因的克隆与组织表达

Molecular Cloning and Expression Profiles of β-actin Gene in Various Tissues of the Horseshoe Crab,Tachypleus tridentatus

中国鲎(Tachypleus tridentatus)是一种古老的海洋动物.利用RT-PCR和RACE等分子生物学技术,获得中国鲎β-肌动蛋白基因(中国鲎β-actin,简称为TTBA)全长cDNA序列,总共1 515bp,包括70bp 5′非编码区(untranslated regions,UTR)、314bp 3′UTR和一个1 131bp的开放阅读框(open reading frame,ORF).ORF可编码376个氨基酸残基,总分子质量约为41 807.7u.同源蛋白的序列比较结果显示,TTBA推导氨基酸序列与其他物种具有很高的相似性,推测β-actin基因具有很高的遗传保守性.而基于β-actin蛋白序列比对而绘制的系统进化树显示中国鲎与其他节肢动物具有较高的相似性,此结果与其现行的分类地位基本一致.以Real-time RT-PCR法检测表明,在卵子发生各期的卵巢组织中TTBA表达量保持稳定(p>0.05),可作为定量检测的内参基因.

The horseshoe crab（Tachypleus tridentatus）is an ancient marine organism.In this study,a full-length cDNA ofβ-actin（designated as TTBA）was cloned from T.tridentatus using RT-PCR and RACE method.Data showed that the full-length cDNA was 1 515 bp,including a 5′untranslated region（UTR）of 70 bp,a 3′UTR of 314 bp,and an open reading frame（ORF）of 1 131 bp nucleotides.The ORF encoded a polypeptide of 376 amino acids with calculated molecular mass of 41 807.7u.Amino acid sequence alignment showed the TTBA had high similarity with that in other organisms.It suggested thatβ-actin was highly conservative.Phylogenetic analysis based on comparison ofβ-actin proteins showed that T.tridentatus was more similar to arthropod than to other invertebrate species.This result was consistent with the current evolution relationship.Moreover,gene expression profiles by Real-Time RT-PCR revealed that the abundance of TTBA mRNA kept steady during oogenesis in ovary（p〉0.05）indicating that TTBAis suitable as an endogenous control for gene expression study.