Expression,Purification And Activation Of The Silkworm(Bombyx Mori) Dorsal

As an important economic insect,silkworm,Bombyx mori,it often causes huge economic loss to sericulturist since silkworms are easily infected by pathogenic microbes during breeding.Hence,it is necessary to study the molecular basis that how the silkworm defenses pathogens thoroughly.Insect relies on innate immune system to defence against pathogens and parasites.The production of antibacterial peptides(AMP)is one main immune responses.AMP is produced through immune deficiency(IMD)and Toll signaling pathways.The Rel/NF-?B transcription factors can act as key regulators to modulate the expression of immune-related genes in response to immune challenge or environmental stress in two pathway.In Drosophila,Dorsal,the Rel/NF-?B transcription factor,which act on the Toll pathway,is activated in response to the infection with fungi and Gram-positive bacteria.Dorsal is localized in the cytoplasm and interact with Cactus in unstimulated cells.In response to an infection,Dorsal translocate into the nucleus by dissociating from Cactus,and activate antifungal peptide genes such as drosomycin.In silkworm,there is no eveidence demonstrate that Dorsal acts on the Toll pathway and it is not clear whether Dorsal enters the nucleus from the cytoplasm after isolation.In this study,we selected silkworm as the model species,cloned Dorsal gene from silkworm,constructed the prokaryotic expression vector,expressed and purified the Dorsal protein and prepared the antibody of the silkworm Dorsal protein.We selected Gram-negative bacteria,Bacillus bombysepticus,and two Gram-positive bacterias,Yersinia pseudotuberculosis and Escherichia coli,to challenge silkworms,then extracted nuclear and cytoplasmic proteins from fat body to detect the content of Dorsal.We also infected silkworms with a gram-negative bacteria Bacillus bombysepticus and a Gram-positive bacteria Escherichia coli to detect whether the changes in the expression levels of Attactin2,Cercropin E,Lebocin,Gloverin3 and Moricin antimicrobial peptides were consistent with the change of Dorsal protein.Our results show the following:(1)We expressed recombinant Dorsal protein of the silkworm in and raised(1)We expressed recombinant Dorsal protein of the silkworm in E.Coli and raised silkworm Dorsal antibody.(2)We detected the Dorsal protein in the cytoplasm and nucleus protein of infected silkworm fat body cells with Dorsal antibody.It was found that Dorsal protein was mainly in the form of dimer in the cytoplasm and not in the nucleus after infected 12 hours.24 hours after infection,the Dorsal in the cytoplasm was actived and changed in protein levels.The partially activated Dorsal protein was transferred from the cytoplasm to the nucleus after infection and the effect of translocation was more apparent when infected with Gram-positive bacteria.(3)Using real-time quantitative PCR we found that the expression levels of Attactin2,Cercropin E,Lebocin,Gloverin3 and Moricin in the silkworm fat body were significantly increased after infection with B.bombysepticus and E.coli,which indicated that bacterial infection activate the expression of antimicrobial peptides.The results showed that Dorsal protein was activated in the silkworm which was infected by bacteria.Dorsal protein changed and part of the activated Dorsal protein transported from the cytoplasm into the nucleus.Moreover,the activation induced by Gram-positive bacteria was more significant than by Gram-negative bacteria.