Nf-κB Mediates The Activation Of Cuticular Protein Gene BmCPT1by LPS In Silkworm,Bombyx Mori

Insect cuticle is an important barrier to separate itself from the outside world, which is also a physical barrier for defending the pathogenic microorganisms. Cuticular proteins and chitin are the most essential ingredients of the cuticle. As the structural proteins, cuticle proteins play important roles in the cuticle. The insect innate immune system can recognize the target molecules on the surface of bacteria via the pattern recongnize receptors (PRRs), which distinguish "self" from the "non-selfs". Insect uses the cellular and humoral immunal reactions of the Toll and IMD pathways, and the ProPo cascade reactions to resist the bacterial infections. NF-κB pathway acts at the downstream of the Toll and IMD signaling pathways which is activated by the stimulus. Being dissociated from the inhibitors, NF-κBs transport into the nucleus quickly and bound to the promoter of the antimicrobial peptide genes. The expressions of these genes are essential for the pathogen clearance. Although the draft sequence of the whole genome of the silkworm (Bombyx mori) has been published in2004, which has greatly promoted the silkworm innate immunity research, but research for understanding the activation of silkworm cuticular protein by the immune signals is still unclear.In the present study, we obtained the sequence of the BmCPT1gene from the silkworm genome database. We cloned the2002bp promoter upstream sequences of this gene and identified a NF-κB binding site in this region by the TESS program. We then used the pGL3-basic vector and constructed a transfection luciferase reporter vector pGL3-basic{pBmCPTl-Luciferase} plasmid. The resultant plasmid was transfected into silkworm ovary cell line BmN. The expressions of luciferase were observed by adding LPS and NF-κB signaling blocker PDTC. The luciferase reporter assay showed that the promoter of the BmCPT1gene was activated by LPS and this activation was inhibited by PDTC. To further verify this result, we injected the dead E. coli into day3of the fifth instar silkworm larvae. After injection for12hours, we extracted nucleoprotein from the fatbody. The labeled probe and mutated probe were designed for the gel-shift assay based on the NF-κB sequence of the BmCPTl promoter. Our research provided the direct evidence of that silkworm NF-κB transcription factor BmRelishl was able to recognize the NF-κB binding site of the BmCPTl promoter and then activate the expression of BmCPTl gene by LPS.