Clone,Expression And Characterization Of Keap1 And Nrf2 From Musca Domestica

Housefly(Musca domestic) feeds saprophages and lives with so much pathogenic bacteria,but it has powerful immune regulation mechanisms to resist bacteria infection.Based on transcriptome database of M. domestic owned by our laboratory and geneome database of M. domestic published by NCBI,we analyzed biological information about nuclear transcription factor Nrf2 and its inhibitor Keap1.Md Keap1 encoded 858 amino acids weighted 95.75 ku and Md Nrf2 encoded 924 amino acids weighted 113.72 ku.Their isoelectric points respectively were 7.68 and 5.73.The secondary and three-dimensional structure predictions confirmed that Keap1 had BTB domain binding with ubiquitin ligating enzyme and six repeat motifs Kelch binding with Nrf2.The main conserved domain of Nrf2 was b Zip to combine with specific DNA sequnece with small Maf together.We obtained recombinant vectors used for prokaryotic expression and antibody preparation.Western Blot detection reflected the expression levels of Keap1 and Nrf2.The m RN A level of Keap1 and Nrf2 were detected by RT-q PCR.The larvas were infected by Staphylococcus aureus and Escherichia coli and gathered after 6 h,12 h,24 h,48 h and 72 h as experimental group.The control group was dealed without any bacteria.The results showed that Keap1 was down-regulated and Nrf2 was opposite.The Keap1 expression level was highest significantly in fat body and lowest in hemolymph.The m RNA expression Nrf2 was highest in the intestinal tract and lowest in the cuticula.Interfering larvas with bacteria expressed double-stranded RN A,we found that the m RNA level of Keap1 and Nrf2 were knockdown indeed and the results were significant.The larva death rate in Keap1 interfered was higher than the control group. In contrast,The larva death rate in Nrf2 interfered was lower than the control group obviously.The activity of three protection enzymes,SOD,C AT,POD were up-regulated under heat shock treatment 15 min,30 min and 1 h.We deduced that higher temperature could lead to oxidative damage.The protein expression of Keap1 was down-regulated and Nrf2 was upregulated.