The Key Laboratory of Mariculture, Ministry of Education, Ocean University of China, Qingdao 266003, China.
The Key Laboratory of Mariculture, Ministry of Education, Ocean University of China, Qingdao 266003, China.
Fish Shellfish Immunol. 2019 Sep;92:489-499. doi: 10.1016/j.fsi.2019.06.006. Epub 2019 Jun 18.
In this study, we cloned the full-length cDNA of the Kelch-like ECH-associated protein 1 (Keap1) from the scallops Chlamys farreri (C. farreri). Sequences alignment and phylogenetic analysis showed that CfKeap1 was highly specific in the scallops, and the amino acid sequence identity value is closer to that in zebrafish Keap1b and Nothobranchius furzeri Keap1b than Keap1a. The highest transcription level of CfKeap1 expression was detected in the digestive glands. The gene expressions of CfKeap1, NF-E2-related nuclear factor 2 (Nrf2), Superoxide Dismutase (SOD), Catalase (CAT) and Glutathione Peroxidase (GPx) in digestive glands were evaluated by quantitative real-time PCR (qRT-PCR) after being exposed to benzo(a)pyrene (BaP) (0.25, 1and 4 μg/L) for 15 days, which indicated that the activation of Nrf2 and Keap1 expression can be significantly induced under BaP exposure. RNA interference (RNAi) experiments were conducted to examine the expression profiles of CfKeap1, Nrf2, antioxidant genes (Cu/Zn-SOD, CAT and GPx), mitogen-activated protein kinase (MAPKs) and protein kinase C (PKC) signaling pathways key genes in digestive glands and gills when exposed to BaP. Results showed that the mRNA level of CfKeap1 was significantly decreased by 60.69% and59.485%. The changes of CfKeap1 and Nrf2 suggested that the enhancement of Keap1 expression stimulating Nrf2 degradation. Furthermore, the expression of antioxidant genes were consistent with the Nrf2 gene, which suggesting that Nrf2-Keap1 signaling pathway is required for the induction of antioxidant genes. Besides, the changes of PKC, c-Jun N-terminal kinase (JNK) and p38 genes expression suggested that PKC and MAPKs signaling pathways played a synergistic role with Nrf2-Keap1 signaling pathway in the anti-oxidative defense system of bivalve molluscs. In conclusion, these data demonstrated that Keap1 can sense nucleophilic or oxidative stress factors to regulate the Nrf2 signaling pathway together with Cul3-based E3 Ubiquitin Ligase (E3), and the Nrf2-Keap1 signaling pathway played an important role in modulating gene expression of antioxidant enzymes in bivalve mollusks.
在这项研究中，我们从扇贝 Chlamys farreri（C. farreri）中克隆了 Kelch-like ECH-associated protein 1（Keap1）的全长 cDNA。序列比对和系统发育分析表明，CfKeap1在扇贝中具有高度特异性，其氨基酸序列同一性值更接近斑马鱼 Keap1b 和 Nothobranchius furzeri Keap1b，而不是 Keap1a。在消化腺中检测到 CfKeap1 表达的转录水平最高。通过定量实时 PCR（qRT-PCR）评估了暴露于苯并（a）芘（BaP）（0.25、1 和 4μg/L）15 天后消化腺中 CfKeap1、NF-E2 相关核因子 2（Nrf2）、超氧化物歧化酶（SOD）、过氧化氢酶（CAT）和谷胱甘肽过氧化物酶（GPx）的基因表达，结果表明，在 BaP 暴露下，Nrf2 和 Keap1 表达的激活可以显著诱导。进行了 RNA 干扰（RNAi）实验，以研究消化腺和鳃中 CfKeap1、Nrf2、抗氧化基因（Cu/Zn-SOD、CAT 和 GPx）、丝裂原活化蛋白激酶（MAPKs）和蛋白激酶 C（PKC）信号通路关键基因的表达谱当暴露于 BaP 时。结果表明，CfKeap1 的 mRNA 水平显著降低了 60.69%和 59.485%。CfKeap1 和 Nrf2 的变化表明，Keap1 表达的增强刺激了 Nrf2 的降解。此外，抗氧化基因的表达与 Nrf2 基因一致，表明 Nrf2-Keap1 信号通路是诱导抗氧化基因所必需的。此外，PKC、c-Jun N-末端激酶（JNK）和 p38 基因表达的变化表明，PKC 和 MAPKs 信号通路与 Nrf2-Keap1 信号通路在双壳类软体动物的抗氧化防御系统中发挥协同作用。总之，这些数据表明，Keap1 可以感知亲核或氧化应激因子，与 Cul3 基 E3 泛素连接酶（E3）一起调节 Nrf2 信号通路，并且 Nrf2-Keap1 信号通路在调节双壳类软体动物抗氧化酶的基因表达中起着重要作用。
