背角无齿蚌σ-GST基因克隆与五氯酚胁迫表达分析

Cloning of σ-GST Gene in Anodonta woodiana and Its Expression Analysis Under Pentachlorophenol Stress

谷胱甘肽S-转移酶(GST)在机体抗击氧化应激中发挥重要作用。我们前期研究表明,五氯苯酚(PCP)处理对背角无齿蚌(Anodonta woodiana)具有显著的氧化应激和急性毒性效应。为了探讨PCP慢性胁迫效应,本研究将背角无齿蚌随机分为对照组和PCP处理组,PCP处理组和对照组分别用13.9 mg/L和相同体积二甲亚砜处理;同时,克隆出σ型谷胱甘肽S-转移酶并命名为σ-GST,分析PCP对σ-GST表达的影响。σ-GST全长cDNA包括132 bp的5’端非编码区,80 bp的3’端非编码区,609bp的开放阅读框(ORF)。开放阅读由203个氨基酸组成的多肽链。σ-GST具有GST的标签序列和保守氨基酸,与其他物种σ类GST序列具有高度同源性。σ-GST广泛表达于斧足、外套膜、闭壳肌、心脏、肝胰腺、血淋巴和鳃。PCP处理后,肝胰腺、鳃和血淋巴中σ-GST表达水平显著增加。与对照组相比,PCP处理后肝胰腺σ-GST mRNA水平第1、3和第15天分别增加了28.73%、70.37%(P<0.05)和6.64倍(P<0.01)。与对照组相比,PCP处理后鳃中σ-GST mRNA水平在整个实验过程中增加了1.14倍。结果表明,背角无齿蚌ρ-GST表达水平上调有助于对抗PCP处理所产生的胁迫效应,提高动物环境耐受能力。

Glutathione S-transferases(GSTs)play a prominent role in protecting cells against oxidative stress. Our previous studies reveal that the pentachlorophenol(PCP)causes significant oxidative stress and acute toxic effects on Anodonta woodiana,but its chronic toxicity remains unclear. In order to investigate the chronic effect of PCP,A. woodiana were randomly grouped into PCP-treated group with13.9 μg/L concentrations of PCP and control group with the same volume of dimethyl sulfoxide. Meanwhile,one GST sequences were cloned from A. woodiana and named as σ-GST,and the effect of PCP on the σ-GST was analyzed. The full-length cDNA of σ-GST consisted of 132 bp5’ untranslated region(UTR),80 bp 3’ UTR and 609 bp open reading frame of an encoded polypeptide by 203 amino acids.σ-GST had the tag sequence of GST and conserved amino acids,and had high homology with σ-like GST sequence of other species. The σ-GST was widely expressed in different tissues such as foot,mantle,adductor muscle,heart,hepatopancreas,hemocytes and gill. PCP treatment resulted in a significant increase of σ-GST expression in the hepatopancreas,gill and hemocytes. In the hepatopancreas,σ-GST m RNA level of PCPtreated group increased more than 28.73% at day 1,then 70.37%(P<0.05)at day 3,reached 6.64 times(P<0.01)at day 15 in contrast to the control group. In the gill,σ-GST mRNA expression showed a up-regulation by 1.14 times(P<0.05)in the PCP-treated group during whole experiment,compared with that of control group. These results indicate that up-regulation of σ-GST expression in A. woodiana is conducive to its resistance to the stress from PCP treatment during experiment.