泥蚶谷胱甘肽过氧化物酶基因的全长克隆与表达分析

FULL-LENGTH CDNA CLONING AND EXPRESSION ANALYSIS OF GLUTATHIONE PEROXIDASE FROM BLOOD CLAM TEGILLARCA GRANOSA

为了探讨谷胱甘肽过氧化物酶(Glutathione peroxidase,GPx)基因在泥蚶应激反应中的作用,研究采用RACE技术克隆了泥蚶GPx基因(TgGPx)c DNA全长,其c DNA全长1195 bp,包含45 bp 5′-UTR,639 bp开放阅读框(ORF)和511 bp 3′-UTR。ORF编码212个氨基酸残基,预测蛋白分子量为24.3 k D,理论等电点为8.33,其中,第53个氨基酸U是由密码子202UGA204编码的硒代半胱氨酸(Se-Cys)。在3′-UTR上存在一段序列,形成一种独特的茎环结构,即SECIS元件。SECIS元件在密码子UGA翻译为Se-Cys的过程中起决定性作用。通过序列比对与系统进化分析,发现软体动物中也存在不同种类的GPx基因,TgGPx与GPx1和GPx2的亲缘关系较近。利用qRT-PCR技术对TgGPx在泥蚶的不同组织以及重金属刺激后的表达量进行分析,结果表明,TgGPx在泥蚶的5个组织中都有表达,但存在组织特异性,在外套膜中的表达量最高,在血细胞中的表达量最低。用重金属铅、铜、镉刺激后,TgGPx在肝胰脏中的表达量显著升高,表明TgGPx在维护机体正常功能方面及泥蚶抵御外界刺激的应激反应中发挥作用。

Glutathione peroxidase（GPx） is an important member of cellular enzymatic antioxidant system regulating stress response of host as an acute protein. In this study, a selenium-dependent glutathione peroxidase（TgGPx） gene from blood clam Tegillarca granosa was cloned and analyzed by rapid amplification of c DNA ends（RACE）. The nucleotide sequence of TgGPx was consisted of 1195 bp with a 45 bp 5′UTR a of 511 bp 3′UTR and 639 bp open reading frame（ORF） encoding a peptide of 212 amino acids with an estimated molecular mass of 24.3 k D and a theoretical isoelectric point of 8.33. TgGPx had a characteristic codon at 202UGA204 that corresponded to Selenocysteine as U53. A selenocysteine insertion sequence（SECIS） element was identified in the 3′-UTR of TgGPx c DNA, which forms a stemloop secondary structure. SECIS element plays a decisive role in the translation of the stop codon UGA into a selenocysteine. Multiple sequence alignment and phylogenetic analysis revealed that there were different types of GPx genes in mollusks. TgGPx has a closer phylogenic relationship with GPx1 and GPx2. TgGPx expressed in all five tissues with the highest m RNA level in mantle and the lowest in haemocytes. The expression pattern of TgGPx in adult may support its role in adult tissue growth and larval development. TgGPx was significantly up-regulated in hepatopancreas by heavy metals（Zn^（2＋）, Cu^（2＋） and Cd^（2＋）） exposure, suggesting TgGPx may play a role in stress response and maintaining the body＇s normal function in T. granosa.