关键词: 家蚕, 免疫负调控, BmCaspase-8-Like（BmCasp8L）, 表达特征, 抗菌肽, BmRelish

Abstract:

【Objective】The objective of this study is to characterize BmCaspase-8-like (BmCasp8L) as an immune negative regulatory molecule in silkworm (Bombyx mori) cell line as well as in B. mori larvae, and to provide a basis for further studies of negative regulation mechanism in insect immunity.【Method】Domain prediction and phylogenetic analysis were performed after cloning of BmCasp8L by RT-PCR. Then fluorescence quantitative PCR was used to investigate the spatial-temporal expression profile of BmCasp8L at different development stages and in different tissues extracted from the 3rd day of 5th-instar larvae and prepupa, as well as the larvae body after bacterial infection. The dsRNA for RNAi was synthesized to silence BmCasp8L in the B. mori larvae, and the effect of BmCasp8L on expression of the anti-microbial peptides was studied. The plasmid for expressing BmCasp8L in cells was constructed. After transfection of BmE cells with the expression constructs or dsRNA, Western blot or quantitative PCR was performed to confirm the over-expression or knock-down of BmCasp8L in cells. Meanwhile, the change in expression of the anti-microbial peptides and cleavage of nuclear transcription factor BmRelish were detected.【Result】BmCasp8L is homologous to Lepidoptera Caspase-6 and mammalian Caspase-8. The similarity between BmCasp8L and N-terminal of BmDredd, DmDredd is 61% and 42%, respectively, but it lacks the C-terminal Caspase domain. Spatial-temporal expression profile showed that in molting larvae BmCasp8L level was higher than in newly exuviated ones, and the BmCasp8L expression level was significantly increased in the prepupa, 7th day of pupa and 1st day of moth stage. The BmCasp8L expression level in the hemocyte was significantly higher than in other tissues in the 3rd day of 5th-instar, but in the prepupa stage, it was mainly expressed in the silk gland. The BmCasp8L expression level in the 3rd day of 5th-instar larvae increased within 1 h post infection of Bacillus bombyseptieus or Serratia marcescens, and then gradually returned to normal. Injection of dsCasp8L or dsEGFP into the 2nd day of 5th-instar larvae, the expression of BmCasp8L was significantly down-regulated in B. mori injected with dsCasp8L, while the expression of antimicrobial peptide BmCecropinA1 was up-regulated after 24 h. Over-expression of BmCasp8L in BmE cells led to a remarkable decrease of the anti-microbial peptide BmCecropinA1. In addition, over-expression of BmCasp8L suppressed the cleavage of the transcription factor BmRelish. Moreover, the expression level of BmCasp8L could be efficiently knocked down by dsRNA, at the same time, the expression level of BmCecropinA1 was significantly up-regulated. 【Conclusion】Phylogenetic analysis, expression features and functional studies in cells as well as in B. mori larvae all indicated that BmCasp8L acts as an immune negative regulatory molecule by suppressing the cleavage of BmRelish and the expression of anti-microbial peptides, thereby negatively regulating the Imd signaling pathway, and down-regulation of BmCasp8L resulted in an increase of anti-microbial peptides which would potentially increase the resistance of B. mori larvae to bacterial infection.

Key words: Bombyx mori, immune negative regulation, BmCaspase-8-Like (BmCasp8L), expression feature, anti-microbial peptide, BmRelish