College of Life Science, Education Ministry Key Laboratory of Poyang Lake Environment and Resource Utilization, Nanchang University, Nanchang 330031, China.
Hydrobiology Protection and Rescue Center of Jiangxi, Nanchang 330029, China.
Gene. 2022 Dec 30;847:146848. doi: 10.1016/j.gene.2022.146848. Epub 2022 Sep 9.
Avian musculoaponeurotic fibrosarcoma (Maf) proteins play an important role in Nrf2/Keap1 signaling pathway, which mainly resist the oxidant stress. The members of sMaf have a high homology basic leucine zipper (bZIP) and lack trans activation domain, and could interact with other transcriptional regulatory factors as a molecular chaperone. In this study, a full-length MafG-like gene was cloned from Procambarus Clarkii, designated as PcMafG-like, which consisted of an ORF length of 246 bp encoding 82 amino acids, a 5' untranslated region (UTR) of 483 bp, and a 3' UTR of 111 bp. The domain of PcMafG-like had a bZIP-Maf domain that binds to DNA. The cDNA sequence of PcMafG-like was 99 % similar to that of Penaeus vannamei. The mRNA of PcMafG-like was expressed in all tested tissues, and the highest expression was in muscle tissue. Under stimulation of Cu and Cd, PcMafG-like was significantly up-regulated in hepatopancreas and gill, and the same result was testified by situ hybridization. The representative antioxidant genes, CAT, GPx and CZ-SOD, were significantly induced by Cu; CAT and GPx was induced by Cd. PcMafG-dsRNA significantly inhibited the expression of these up-regulated genes, but also inhibited the expression of other detected genes CZ-SOD, GST-θ and GST-1like. The antioxidant effect of PcMafG-like was further verified by oxidative stress markers (T-SOD, CuZnSOD, GPx, CAT, GSH and MDA) kits. Cu and Cd could induce the contents of these oxidative stress markers (MDA, GSH, CZ-SOD, CAT in Cu/Cd treated group, and GSH-Px in Cd group), while interference of PcMafG-like significantly inhibited the up-regulation. Furthermore, hematoxylin-eosin staining experiments showed that the degree of pathological damage was dose-dependent and time-dependent, and the pathological damage was more serious after dsRNA interfered with PcMafG-like. In addition, subcellular localization showed that PcMafG-like gene existed in nucleus. The recombinant protein PcMafG-like was expressed and purified in prokaryotic expression. The affinity analysis of promoter by agarose gel electrophoresis suggested that PcMafG-like could bind with CAT promoter in vitro. This indicated that PcMafG-like could activate antioxidant genes.
禽类肌肉腱膜纤维肉瘤（Maf）蛋白在 Nrf2/Keap1 信号通路中发挥重要作用，主要抵抗氧化剂应激。sMaf 成员具有高度同源的碱性亮氨酸拉链（bZIP）结构，缺乏转录激活结构域，可作为分子伴侣与其他转录调控因子相互作用。本研究从克氏原螯虾中克隆了全长 MafG 样基因，命名为 PcMafG-like，该基因包含一个 246bp 的开放阅读框，编码 82 个氨基酸，5'非翻译区（UTR）为 483bp，3'UTR 为 111bp。PcMafG-like 的结构域具有与 DNA 结合的 bZIP-Maf 结构域。PcMafG-like 的 cDNA 序列与凡纳滨对虾的相似度为 99%。PcMafG-like 的 mRNA 在所有检测组织中均有表达，在肌肉组织中表达量最高。在 Cu 和 Cd 刺激下，PcMafG-like 在肝胰腺和鳃中显著上调，原位杂交试验得到了相同的结果。代表性抗氧化基因 CAT、GPx 和 CZ-SOD 被 Cu 显著诱导；CAT 和 GPx 被 Cd 诱导。PcMafG-dsRNA 显著抑制这些上调基因的表达，但也抑制了其他检测基因 CZ-SOD、GST-θ 和 GST-1-like 的表达。PcMafG-like 的抗氧化作用进一步通过氧化应激标志物（T-SOD、CuZnSOD、GPx、CAT、GSH 和 MDA）试剂盒得到验证。Cu 和 Cd 可以诱导这些氧化应激标志物（MDA、GSH、CZ-SOD、CAT 在 Cu/Cd 处理组，以及 Cd 组中的 GSH-Px）的含量增加，而 PcMafG-like 的干扰显著抑制了它们的上调。此外，苏木精-伊红染色实验表明，病理损伤程度呈剂量依赖性和时间依赖性，dsRNA 干扰 PcMafG-like 后病理损伤更严重。此外，亚细胞定位显示 PcMafG-like 基因存在于细胞核中。在原核表达系统中表达并纯化了重组蛋白 PcMafG-like。琼脂糖凝胶电泳的启动子亲和分析表明，PcMafG-like 可以在体外与 CAT 启动子结合。这表明 PcMafG-like 可以激活抗氧化基因。
