Cloning and Functional Analysis of CncC and Keap1 Genes in Silkworm

CncC/keap1-ARE is an important signaling pathway for detoxification and antioxidation in Diptera and Coleoptera insects. However, such a signaling pathway has not been studied in Bombyx mori. In this study, BmCncC and Bmkeap1 genes were cloned, their amino acid sequences were analyzed, and each functional domain was mapped. Through phylogenetic analysis and sequence comparison among multiple species, we found that the Neh1 motif of CncC was highly conserved and the DLG motif was replaced by the DMG motif in Neh2. Conformational analysis showed that Neh1 of BmCncC forms a hairpin structure to bind DNA. The DGR region of Bmkeap1 contained abundant beta sheets, which was involved in the recognition of Neh2. The transcription and expression analyses showed that both BmCncC and Bmkeap1 were highly expressed in the first instar larvae, and these two genes were expressed at a high level in the reproductive gland, fat body, and head. The transcriptional and expression levels of Akt and BmCncC in the fat body were significantly upregulated, and the expression of Bmkeap1 was downregulated after the phoxim treatment in silkworm. The transcriptional levels of CncC-regulated detoxification enzymes GST, cyp4M5, cyp6AE2, and cyp9G3 were increased by 4.026-, 5.246-, 3.821-, and 9.787-fold, respectively, while the activities of GST and CYP450 were increased by 1.521- and 1.231-fold, respectively, after phoxim treatment. These results indicated that the BmCncC/Bmkeap1 signaling pathway was activated by phoxim, leading to the expression of downstream detoxifying enzymes and detoxification of phoxim in silkworm.