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

发布时间：2018-05-21 01:31

本文选题：草鱼 + 核因子E相关因子(Nrf)　；参考：《水产学报》2018年02期

【摘要】：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Neh[Nrf2-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。
[Abstract]:A large amount of reactive oxygen species can be produced during respiratory burst, in which nuclear factor E2 related factor 2nrf2 plays an important role. The Nrf2 gene of grass carp was cloned and analyzed, then the polyclonal antibody of Nrf2 protein was prepared, and the relationship between the polyclonal antibody and respiratory burst was studied. The cDNA length of grass carp Nrf2 gene is 1994 BP, and the open reading frame is 1782 BP, encoding 593 amino acids. Amino acid sequence alignment showed that the Nrf2 gene of grass carp had the highest homology with common carp (87%), and the Nrf2 gene of grass carp contained six conserved Neh [Nrf2-Epoxy chloropropane(ECH)homology] regions during evolution. The results of real-time quantitative PCR(q RT-PCR and Western blotanalysis showed that the total antioxidant activity of Nrf2 gene in 8 tissues of grass carp was significantly increased after treated with tert-Nrf2 activator, tert Ding Ji hydroquinone (t BHQ). The positive oxygen species (Ros) down-regulated the m RNA expression of nrf2 and its downstream HO-1 and GST genes. Western blot and immunofluorescence assays showed that the protein expression of nrf2 was also up-regulated after CIK treatment with t BHQ, and accompanied by the phenomenon of nuclear entry. The study shows that the conserved grass carp Nrf2 gene is widely expressed in the body and can regulate the respiratory burst by up-regulating the expression of antioxidant genes and down-regulating the production of reactive oxygen species (Ros).
【作者单位】：华中农业大学水产学院淡水水产健康养殖湖北省协同创新中心池塘健康养殖湖北省工程实验室农业部淡水生物繁育重点实验室;仲恺农业工程学院动物科技学院广州市水产病害与水禽养殖重点实验室;
【基金】：国家自然科学基金(31502199,31572657,31372563) 广东省海洋与渔业局基金(A201512C003,2015-115) 湖北省科技支撑计划(2015BBA228,YSF2015000255) 武汉市科技局资助课题(2016020101010089) 中央高校基本科研业务费专项(2014PY035)~~
【分类号】：Q78;S917.4

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