草鱼核因子E2相关因子2基因克隆分析及其对呼吸爆发的调控作用

呼吸爆发过程可以产生大量的活性氧,核因子E2相关因子2(Nrf2)在此过程中起着重要作用。实验克隆和分析了草鱼的Nrf2基因,随后制备了Nrf2蛋白的多克隆抗体,研究了其和呼吸爆发的关系。草鱼Nrf2基因cDNA长度为1994 bp,开放阅读框为1782 bp,编码593个氨基酸(aa)。氨基酸序列比对发现,草鱼Nrf2基因与鲤的同源性最高,为87%;草鱼Nrf2基因含有6个进化过程中保守的NehNrf2-Epoxy chloropropane(ECH)homology]区。实时定量PCR(q RT-PCR)和蛋白印迹法(Western blot)检测结果表明,Nrf2基因在检测的草鱼8个组织中均有表达。Nrf2激活剂叔丁基对苯二酚(t BHQ)处理草鱼肾细胞(CIK)后,CIK细胞总抗氧化能力显著上调,产生的活性氧下调,Nrf2及其下游的HO-1和GST基因的m RNA表达上调。Western blot和免疫荧光(IF)检测结果表明,t BHQ处理CIK后,Nrf2的蛋白表达也上调,并伴随着入细胞核现象。研究表明,保守的草鱼Nrf2基因在机体中广泛表达,能通过上调自身及其下游抗氧化基因的表达下调细胞活性氧的产生,从而参与调控呼吸爆发过程。

Cloning and analysis of nuclear factor E2-related factor 2 (Nrf2) and its function in the regulation of respiratory burst in grass carp (Ctenopharyngodon idella)

Nuclear factor E2-related factor 2 (Nrf2) plays a key role in the regulation of respiratory burst which can produce amount of reactive oxygen species (ROS). In the present report, we have cloned and analyzed the Nrf2 gene from grass carp (Ctenopharyngodon idellus), generated its polyclonal antibody, and characterized its role in regulation of ROS. The obtained cDNA of Nrf2 gene was 1994 bp, containing an open reading frame with 1782 bp encoding 593 amino acids. The amino acid sequences of Nrf2 from grass carp were highly identical with those from common carp (Cyprinus carpio) with 87 % similarity. Nrf2 from grass carp contained six typical Neh (Nrf2-Epoxy chloropropane (ECH) homology) domains, indicating it was highly conserved in evolution. The qRT-PCR and Western Blot analysis showed that Nrf2 was expressed in all eight detected tissues. The total antioxidant capacity of CIKs were up-regulated but ROS generated from CIK cells were down-regulated and the mRNAs of Nrf2 and its downstream gene (GST and HO-1) were all up-regulated when CIK was exposed to the ROS inducer, tert-Butylhydroquinone (tBHQ). Similarly, the expression of Nrf2 protein was also increased and it was translocated in the nucleus of CIK exposed to tBHQ. In conclusion, evolutionary conserved Nrf2 was ubiquitously expressed in the tissues of grass carp and it involved in down-regulating the generation of ROS in CIK cells via up-regulating the expression of itself and its downstream antioxidant genes, so as to regulate respiratory burst.