斑节对虾含硒谷胱甘肽过氧化物酶基因全长克隆及表达分析

Full-length cDNA cloning and expression analysis of selenium-dependent glutathione peroxidase from Penaeus monodon

为了研究含硒谷胱甘肽过氧化物酶(selenium-dependent glutathione peroxidase,SeGPx)在斑节对虾应激反应和卵巢发育中的作用,实验以斑节对虾肝胰腺转录组数据中筛选获得的Se-GPx基因(Pm Se-GPx)片段为基础,利用RACE技术获得Pm Se-GPx基因的c DNA全长,并对其进行了生物信息学分析;利用荧光定量PCR技术研究了Pm Se-GPx在斑节对虾不同组织、卵巢发育各期、肝胰腺组织在pH9、硫酸铜(Cu^(2+))应激下的相对表达量和变化趋势。结果显示,Pm Se-GPx基因c DNA全长为959 bp,5'非编码区(UTR)长10bp,开放阅读框(ORF)长639 bp,编码212个氨基酸,310 bp的3'UTR包含一个硒代半胱氨酸插入序列(SECIS),ORF中的密码子209TGA211编码硒代半胱氨酸(selenocysteine,U67)。实时定量PCR实验结果表明,Pm Se-GPx在斑节对虾的肝胰腺、卵巢、精巢、心脏等组织中均有表达,其中肝胰腺中表达量显著高于其他组织,卵巢次之;卵巢发育阶段表达结果则显示Pm Se-GPx在卵巢发育各期均有表达,在卵巢Ⅲ期表达量最高,其次是V期;在Cu^(2+)胁迫下,其表达量总体呈现先下降后上升然后又下降的趋势;在环境pH为9胁迫下,表达趋势呈现先下降,后回升,然后又下降,再大幅上升的趋势。研究表明,SeGPx在斑节对虾卵巢发育过程中具有重要作用,且Se-GPx参与斑节对虾对环境理化因子应激的免疫调控。

Selenium-dependent glutathione peroxidase（denoted as Se-GPx）plays an important role in the enzymatic antioxidant defense system. It clears the reactive oxygen species（ROS） to avoid the cellular damage. In order to study the function of Se-GPx in immune system and the ovarian development of Penaeus monodon, the full-length cDNA sequence of Se-GPx from black tiger shrimps（denoted as Pm Se-GPx） was obtained by high-throughput transcriptome sequencing and RACE. The full-length c DNA of Pm Se-GPx is 959 bp, including an open reading frame（ORF） of 639 bp encoding a polypeptide of 212 amino acids, a 5＇UTR of 10 bp and a 3＇UTR of 310 bp containing a conserved selenocysteine insertion sequence（SECIS） element. The molecular mass of the deduced amino acid（aa） sequence was 23.65 k Da with an estimated p I of 4.61. Blast and phylogenetic analyses suggest that Pm Se-GPx is a new member of the shrimp GPxs family. The tertiary structure of Pm Se-GPx protein presents like a bag and there is a catalysis site, which contains couplet of four conservative residues（cysteine C/selenocysteine U, glutamine Q, tryptophan W and asparagine N）. Pm Se-GPx m RNA expression was detected in nine tissues by real-time PCR. Pm Se-GPx m RNA expressed was the highest in the hepatopancreas and moderate in the ovary.The result showed that Pm Se-GPx was expression mainly in hepatopancreas which plays an important role in detoxification. At different stages of ovary development, Pm Se-GPx was expressed highly at StageⅢ and Stage V, which indicated a large number of ROS may be produced during these development stages. Pm Se-GPx may protect the ovary from the oxidization damage. Under the stress of Cu^（2＋）, the expression of Pm Se-GPx showed a trend of decline, but recovered at 48 h, and then decreased. The result showed that the stress of Cu^（2＋） may inhibit the expression of Pm Se-GPx. Under the stress of pH 9, expression trend of Pm Se-GPx first declined, but recovered at 24 h, then rose significantly at the 96 h. The result showed that neutral environment may inhibit the expression of Pm Se-GPx but aqueous alkaline medium may promote it. Presumably, Se-GPx plays an important role during the ovarian development and immune regulation of P. monodon. Our study provides a basis for further research in reproduction and immunology of P. monodon.