BmBrC-Zs Mediates The Activation Of BmAMPs By 20E Ininnate Immune System Of Silkworm,Bombyx Mori

Insects possess an efficient innate immune system to protect themselves from pathogens and parasites,which includes the humoral and the cellular immune system.Antimicrobial peptides(AMPs)are important effectors in the humoral immune response of insects.AMPs play an important role in prevention of invasion from foreign microorganisms through inhibiting the proliferation of invasive microorganisms or even killing them directly.Previous studies have shown that 20-hydroxyecdysone(20E)plays an important role in regulating the innate immune response of insects.20 E can up-regulate the expression of immune-related genes such as AMPs in insects through Immune Deficiency(IMD)signaling pathway when they are invaded by pathogens.However,it remains unclear how 20 E regulates the expression of AMPs.In this paper,we investigated whether the transcription factorBmBrC-Zs regulate BmAMPs induction by 20 E in humoral immune system of the domestic silkworm,Bombyx mori.Our results confirmed that BmBrC-Zs did mediate the up-regulation expression of BmAMPs by 20 E.The main results are as follows:1.Gram-negative Escherichia.coli and Gram-positive Bacillus.bombyseptieus were injected into day 3 larvae of the fifth instar silkworms.The survival rate of silkworms was significantly increased and the number of colonies was significantly decreased when 20 E was injected before pathogen invading.These results indicated that 20 E can promote the immune response of the domestic silkworm.2.The fat boy,which secrets AMPs,were induced by 20 E in both vivo and vitro,and the expression of 7 BmAMPs were surveyed.It was found that 20 E up-regulated the expression of BmAMPs in the fat boy,and the expression levels of different BmAMPs were significantly different.BmCeCE showed the highest expression level so that it was selected as one of main target genes in our research.3.The TFBS in the promoter region of 11 BmAMPs genes were predicted,BrC-Z elements were predicted in 10 of 11 BmAMPs,indicated BmBrC-Zs may regulate 20 E activation of BmAMPs.The expression level of 7 BmAMPs genes were detected after BmBrC-Zs overexpression in BmNs cell line,BmLeB3 andBmGlv2 showed the highest expression level so that chosen as another two target genes in our research.4.The luciferase reporter vectors of BmCeCE,BmGlv2 and BmLeB3 promoters were constructed and transfected into the BmNs cell line.It was confirmed that these three BmAMPs genes were up-regulated by 20 E signal.Then a mutant of BrC-Z elememt in BmLeB3 was made and the activity of BmLeB3 mutant promoter was significantly reduced and no longer subject to 20 E regulation after mutation,indicating that BrC-Z is an important regulator in up-regulation of BmAMPs by 20 E.5.The BmAMPs promoter activity was significantly increased in the BmNs cell line after BmBrC-Zs overexpression,proving that BmBrC-Zs regulate up-regulation of BmAMPs by 20 E more directly.In summary,in the humoral immune system of the silkworm,20 E signal can up-regulate the expression of BmCeCE,BmGlv2 and BmLeB3,this process was confirmed to be regulated by BmBrC-Zs transcription factors in this paper.