背角无齿蚌硒谷胱甘肽过氧化物酶基因的克隆及2,4-二氯苯酚、2,4,6-三氯苯酚和五氯苯酚对其表达的影响

Cloning of AwSeGPx from Anodonta woodiana and Effects of 2,4-dichloro-phenol,2,4,6-trichlorophenol and Pentachlorophenol on Its Transcription

硒谷胱甘肽过氧化物酶(SeGPx)是一类广泛存在于生物体内的重要抗氧化酶,SeGPx可将有毒的过氧化物还原成无毒的羟基化合物,从而保护细胞膜结构及功能不受过氧化物干扰和损害。为了探讨2,4-二氯苯酚(2,4-DCP)、2,4,6-三氯苯酚(2,4,6-TCP)和五氯苯酚(PCP)对背角无齿蚌(Anodonta woodiana)的胁迫效应,本研究克隆出AwSeGPx全基因序列,分析2,4-DCP、2,4,6-TCP和PCP对AwSeGPx表达的影响。AwSeGPx的cDNA序列全长870 bp,开放阅读框为585 bp,编码195个氨基酸。推导的AwSeGPx蛋白序列包括GPx家族标签序列(68LGFPCNQF75)和活性位点序列(156WNFEKF161)、一个典型终止密码子(165TGA167)编码的硒代半胱氨酸(U^(44))、酶催化活性相关保守氨基酸位点:谷氨酰胺(Q^(74))、精氨酸(R^(94)和R^(151))和色氨酸(W^(156))。在相同浓度条件下,PCP对背角无齿蚌毒性效应大于2,4-DCP和2,4,6-TCP。与对照组相比,浓度60、120、240、480和960μg·L^(-1)的2,4-DCP处理后肝胰腺AwSeGPx mRNA水平增加了1.79倍以上(P<0.05);浓度100、200、400和800μg·L^(-1)的2,4,6-TCP处理后AwSeGPx mRNA水平增加了1.01倍以上(P<0.05);浓度20、40、80、160和320μg·L^(-1) PCP处理能够显著诱导AwSeGPx的表达。以上结果表明,2,4-DCP、2,4,6-TCP和PCP处理对背角无齿蚌肝胰腺中AwSeGPx表达具有明显的诱导作用,这种诱导效应与动物提高过氧化物的还原能力和增强环境的耐受能力密切相关。

Selenium dependent glutathione peroxidase(SeGPx)belongs to the family of selenoprotein,which acts mainly as an antioxidant role in the cellular defense system.In order to explore the effect of 2,4-dichlorophenol(2,4-DCP),2,4,6-trichlorophenol(2,4,6-TCP)and pentachlorophenol(PCP)on the freshwater bivalve Anodonta woodiana,a SeGPx full length cDNA was cloned from A.woodiana and named as AwSeGPx,and its expressions were analyzed by real-time PCR.The full length cDNA consists of 870 bp,an open reading frame(ORF)of 585 bp encoded a polypeptide of 195 amino in which conserved domain(68LGFPCNQF75)and a GPx active site(156WNFEKF160)were observed.It had a characteristic codon at 165TGA167 that corresponds to selenocysteine(Sec)amino acid as U^(44).The catalytically related amino acid residues consisting of Gln(Q^(74)),Arg(R^(94)),Arg(R^(151))and Trp(W^(156))were identified in the deduced AwSeGPx protein sequence.Under similar condition,clams A.woodiana showed more sensitive to PCP than 2,4-DCP and 2,4,6-TCP.In the 2,4-DCP treated groups(60,120,240,480 and 960μg·L^(-1)),AwSeGPx mRNA level of hepatopancreas increased more than 1.79 times(P<0.05)compared with that of control group.In the 2,4,6-TCP treated groups(100,200,400 and 800μg·L^(-1)),AwSeGPx mRNA level of hepatopancreas increased more than 1.01 times(P<0.05).In the PCP treated groups(20,40,80,160 and 320μg·L^(-1)),expressions of AwSeGPx were significantly induced compared with that of control group in the hepatopancreas.These results indicate that AwSeGPx plays an active role in inhibiting oxidative stress derived from 2,4-DCP,2,4,6-TCP and PCP treatment in the hepatopancreas.