Cloning And Functional Analysis Of Key Genes In CncC/keap1-ARE Pathway,Bombyx Mori

Silkworm is an important silk insects,but also an important model insect of lepidoptera.Due to the widespread use of chemical pesticides in agriculture and forestry production,which resulted in contaminated mulberry silkworm poisoning and seriously affected the healthy development of the sericulture.CncC/keap1-ARE is an important signal transduction pathway involved in insect antioxidant and detoxification,which had been studied in Drosophila melanogaster and Tribolium confusum.In this study,the CncC and Keap1 genes were cloned and their amino acid sequences were analyzed.Through phylogenetic tree analysis and multi species sequence comparison,we found that the Neh1 motif of CncC was highly conserved in all species.DLG motifs of Neh2 in mammals was instead by DMG motif in Lepidoptera.The Cullin3 binding site and the DGR region of Keap1 were identified.These two sites played an important role in the ubiquitination of Keap1 regulated CncC.The expression characteristics of CncC and Keap1 were highly expressed in the first instar larvae,and the expression of CncC and Keap1 was higher in the gonad,metabolic and neural tissues.The highest expression of the two genes in the nervous system was the main reasons for the high expression of CncC and Keap1 in the first instar silkworm.The CncC and Keap1 transcription levels were significantly up-regulated 29.664-fold and 25.479-fold after 24 hr phoxim treatment was detected by qRT-PCR.The analysis of gene expression profile of the fat body of Bombyx mori.showed that the gene of thioredoxin reductase and glutathione S-transferase gene increased by 1.365-fold,1.335-fold,1.642-fold and 1.765-fold.The superoxide dismutase?SOD?,catalase?CAT?,glutathione S-transferase Delta?GSTd?and thioredoxin reductase?TPx?were up 1.365-fold,1.335-fold,1.642-fold and 1.765-fold in phoxim group.Quantitative real time polymerase chain reaction?qRT-PCR?was used to verify the transcriptional expression of SOD,CAT,GSTd and TPx genes,and the results were consistent with the transcriptome data.The levels of reactive oxygen species?ROS?,malondialdehyde?MDA?and hydrogen peroxide?H₂O₂?were measured by 1.598-fold,1.946-fold and 1.506-fold,respectively.At the same time,the transcriptional level of CYP450 gene?cyp4m5,cyp6ab4,cyp4l6,cyp6ae2,cyp6a8,cyp9g3 and cyp306?which regulated by CncC,was found to be significantly up-regulated after treatment with phoxim in CYP450.The experimental results showed that the treatment of phoxim induced oxidative stress,the expression of CncC was up-regulated,and the transcriptional level of detoxification enzymes and antioxidant enzymes were up-regulated.Nanoparticles TiO₂(TiO₂ NPs),as one of the most important nano materials,was widely used in the field of biology.Previous studies had shown that TiO₂ NPs could effectively slow down the oxidative stress induced by phoxim in Bombyx mori.TiO₂ NPs can effectively reduce the damage of organophosphorus pesticides in the silk gland and nerve tissue.Fat body is an important metabolic detoxification organ in the silkworm,but TiO₂ NPs treatment would affect the metabolism of pesticides in the fat body of Bombyx mori had not been reported.In this study,the SOD,CAT and GSTd transcription levels of the silkworm with TiO₂ NPs treatment were decreased.The levels of ROS,MDA and H₂O₂were restored to normal level with TiO₂ NPs pretreatment and after 24 hr phoxim treatment.The results showed that TiO₂ NPs could alleviate the oxidative stress induced by phoxim.Further elucidate the mechanisms of TiO₂ NPs+phoxim groups,transcription level of P450gene family were detected.The results showed that the expression of cyp4m5,cyp6ab4,cyp6a8 and cyp9g3 in TiO₂ NPs+phoxim group were increased by 2.784-fold,3.047-fold,2.254-fold and 4.253-fold.The high expression of P450 family genes that improve the ability of body fat metabolism of phoxim.TiO₂ NPs was able to induce the expression of some antioxidant genes in the fat body of Bombyx mori,and to alleviate the accumulation of superoxide caused by phoxim.At the same time,TiO₂ NPs treatment could effectively alleviate the oxidative stress caused by phoxim poisoning.In this study,the full-length sequences of CncC and Keap1 were cloned and the basic functional domains of two genes were analyzed.It found that the oxidative stress induced by CncC could be induced by the treatment of phoxim,which induced the expression of detoxification enzymes and antioxidant genes regulated by CncC downstream.TiO₂ NPs pretreatment could effectively alleviate the oxidative stress caused by phoxim in Bombyx mori.The regulation mechanism of CncC/keap1 signaling pathway in the fat body of Bombyx mori was clarified.