斑节对虾GPx3a基因的分子克隆及表达分析

Molecular cloning and expression analysis of glutathione peroxidase 3a gene from black tiger shrimp(Penaeus monodon)

谷胱甘肽过氧化物酶(Glutathione peroxidase,GPx)是生物体内重要的抗氧化酶,能防止过氧化氢对生物体的氧化应激。该研究利用RACE技术获得了斑节对虾(Penaeus monodon)GPx3a(PmGPx3a)的全长c DNA序列,进行了相关生物信息学分析。作者利用荧光定量PCR方法研究了PmGPx3a在斑节对虾不同组织的表达情况。探究了PmGPx3a在不同胁迫条件下(盐度、重金属和细菌)的表达情况。结果表明PmGPx3a c DNA全长1135 bp,其中开放阅读框(ORF)长651 bp,预测编码216个氨基酸。PmGPx3a推导的氨基酸序列与其他动物的GPx3a氨基酸序列具有高度一致性。实时定量PCR结果显示,在高低盐胁迫下,PmGPx3a在肝胰腺中相对表达量都为上升的(p<0.05)。在铜、锌、铬胁迫中,鳃中的PmGPx3a的相对表达量总体呈现下降趋势,在肝胰腺中呈现上升趋势。在哈维弧菌(Vibrio harveyi)刺激下,PmGPx3a在血淋巴中的相对表达量总体呈现上升趋势,24 h后表达量最大,显著高于对照组2.2倍(P<0.05)。以上研究结果表明,PmGPx3a基因参与了斑节对虾对环境胁迫和氧化应激的适应性反应。

Glutathione peroxidase （GPx） is an antioxidant enzyme that plays an important role in protection against oxidative stress by scavenging hydrogen peroxide （H2O2）. The full-length cDNA sequence of PmGPx3a from Penaeus monodon was obtained by high-throughput transcriptome sequencing and rapid amplification of cDNA ends. On this basis, the expression levels of PmGPx3a in different tissues under osmotic stress, heavy metal expo-sure, and bacterial infection were detected by fluorescence-quantitative real-time PCR. The cDNA of PmGPx3a was 1135 bp in length, including an open reading frame （ORF） of 651 bp encoding 216 amino acids. The blast analysis demonstrated that PmGPx3a shared high identity with glutathione peroxidase of Metapenaeus ensis. In the salinity stress experiment, the relative expression of PmGPx3a was found to be upregulated in the hepatopancreas under high salinity and low salinity （P〈0.05）. Under heavy metal exposure, the expression level of PmGPx3a was downregulated in the gill, whereas in the hepatopancreas, the transcript level of PmGPx3a was upregulated （P〈0.05）. After the injection of Vibrio harveyi, the expression level of PmGPx3a was increased up to 2.2-fold compared to that in the PBS group. These results reveal that PmGPx3a plays a significant role in the adaptive response to conditions of oxidative stress and environmental toxicity.