MafG-like contribute to copper and cadmium induced antioxidant response by regulating antioxidant enzyme in Procambarus clarkii.

ABSTRACT

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 Cu2+ and Cd2+, 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 Cu2+; CAT and GPx was induced by Cd2+. 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. Cu2+ and Cd2+ could induce the contents of these oxidative stress markers (MDA, GSH, CZ-SOD, CAT in Cu2+/Cd2+ treated group, and GSH-Px in Cd2+ 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.