虾夷马粪海胆谷胱甘肽过氧化物酶、谷胱甘肽硫转移酶基因的克隆与表达分析

Molecular Cloning and Expression of Glutathione Peroxidase and Glutathione S-transferases Genes From Sea Vrchin （Strongylocentrotus intermedius ）

采用同源克隆方法，结合cDNA末端快速扩增（RACE）技术，从虾夷马粪海胆中克隆到谷胱甘肽过氧化物酶、谷胱甘肽硫转移酶基因的全长cDNA序列，并对其基因结构进行了分析。同时用实时定量PCR方法对这2个基因在脂多糖（I。PS）刺激后不同时间的表达差异情况进行了研究。谷胱甘肽过氧化物酶基因（GPX）的cDNA全长1042bp，其中开放间读框含有657bp，编码219个氨基酸残基，无跨膜结构，无信号肽，为胞内蛋白。实时定量PCR检测发现GPX基因在LPS刺激9h时，表达量最高为对照组的4．47倍（P〈0．01），刺激32h时，表达量恢复到刺激前水平。谷胱甘肽硫转移酶基因（GST）的cDNA全长1931bp，其中开放阅读框含有684bp，编码228个氨基酸残基，无跨膜结构，无信号肽，为胞内蛋白。实时定量PCR检测发现GST基因经LPS刺激8h时表达量最高，48h后表达量恢复到刺激前水平。结果表明，谷胱甘肽过氧化物酶基因GPX、谷胱甘肽硫转移酶基因GST在虾夷马粪海胆免疫方面起到了重要的作用，为深入研究海胆免疫防御机制，改良种质和培育抗病品系，奠定了基础。

In this study , homology-base technique and RACE technique were used to clone Glutathione peroxidase and Glutathione S-transferases genes in Strongylocentrotus intermedius and the stucture of these genes were analysized. Glutathione peroxidase was the intracellular proteins which full-length cDNA was 1042bp with an ORF of 657bp encoding 219 amino acids without transmembrane domain and signal peptide. There were three casein kinase II phosphorylation sites, a glycosaminoglycan site , five protease C phosphorylation sites and two N-meat funny Nichole acylation site. The RT-PCR detection discovered higher-level of GPX mRNA expression after different time of LPS stimulation. The highest GPX expression level, was recorded 9 hours after LPS stimulation in the sea urchin, starvation for 32 hours, the expression returned to the normal levels. Glutathione S-transferases was the intracellular proteins which fulllength cDNA was 1931bp with an ORF of 684bp encoding 228 amino acids without transmembrane domain and signal peptide. There were four casein kinase II phosphorylation sites, an N-glycosylation site, a protease C phosphorylation site and an N-meat funny Nichole acylation site. The highest expression level,of GST was recorded 12 hours after LPS stimulation in the sea urchin, starvation 36 hours, the expression returned to the normal levels. The result indicated that GPX and GST were constitutive and inducible acutephase protein and could play an important role in the immune responses.