Abstract: 【Objective】The objective of this study is to characterize BmFAF as an immune negative regulatory molecule in silkworm cell line as well as in silkworm larvae and provide a basis for further studies of immune homeostasis in insects.【Method】Domain prediction and analysis were performed after cloning of BmFAF by RT-PCR. Then fluorescence quantitative PCR was used to investigate the spatial-temporal expression profile of BmFAF in different developmental stages and in different tissues extracted from the 5th-instar day-3 larvae, as well as the microbe-induced expression pattern after infection. The plasmid for expressing BmFAF in cells was constructed, and dsRNA for RNAi was also synthesized. After transfection of BmE cells with the constructs or dsRNA, Western blotting or quantitative PCR was used to confirm the over-expression or knock-down of BmFAF in cells. Meanwhile, the change in expression of anti-microbial peptides was detected. Finally, BmFAF was partially silenced by dsRNA injected into the silkworm larvae, and the effect of BmFAF on expression of anti-microbial peptides and survival rate of silkworm larvae after bacterial infection was studied.【Result】BmFAF is highly homologous to FAF family members from other species, the similarity between BmFAF and human HsUBXD8, HsFAF1 or Drosophila DmCaspar is 60%, 42% or 47%, respectively. It also contains a conserved UBX domain. Spatio-temporal expression profile showed that except in gonads, the level of BmFAF was slightly higher in immune tissues than in other tissues, and in molting larvae BmFAF level was significantly higher than in newly exuviated ones, particularly in the 2nd molt and 3rd molt stage. In addition, BmFAF expression level was relatively low in the 5th instar, but increased in pre-pupa stage. BmFAF expression level in fat body drastically decreased 0.5 h post infection of Bacillus bombyseptieus or Serratia arcescens, and did not recover within 12 h, whereas BmCecropinA1 was continuously up-regulated and reached the highest degree at 12 h. Over-expression of BmFAF in BmE cells led to a remarkable decrease of anti-microbial peptides, including BmCecropinA1, BmAttacin and BmMoricin. Moreover, the expression level of BmFAF could be efficiently knocked down to 50%-60% by dsRNA, at the same time, the expression level of BmCecropinA1 and BmMoricin was significantly up-regulated. Injection of dsFAF into the 5th-instar day-2 larvae resulted in a decrease of BmFAF to 45% of the level in larvae injected with dsEGFP at 24 h post injection, and the expression level of BmCecropinA1, BmAttacin and BmMoricin was significantly increased. Statistical analysis showed that the survival rate of silkworm larvae injected with dsFAF was higher than the control at 24 h post infection of S. arcescens, at 72 h 40% dsFAF-injected larvae were still alive but no control survived. 【Conclusion】 Domain analysis, expression features and functional studies in cells as well as in silkworm larvae all indicated that BmFAF acts as an immune negative regulatory molecule by suppressing the expression of anti-microbial peptides, and down-regulation of BmFAF resulted in an increase of anti-microbial peptides which would potentially increase the resistance of silkworm larvae to bacterial infection.

Key words: Bombyx mori , immune negative regulation , BmFAF , expression profile , anti-microbial peptide , survival rate