中国农业科学Issue(15)：3085-3093,9.DOI:10.3864/j.issn.0578-1752.2014.15.018
家蚕免疫负调控分子BmFAF的功能
Functional Characterization ofBmFAF as an Immune Negative Regulatory Molecule in Silkworm (Bombyx mori)
摘要
Abstract
[Objective]The objective of this study is to characterizeBmFAF 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 ofBmFAFby RT-PCR. Then fluorescence quantitative PCR was used to investigate the spatial-temporal expression profile ofBmFAF 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 ofBmFAF 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 ofBmFAF 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 orDrosophilaDmCaspar is 60%, 42% or 47%, respectively. It also contains a conserved UBX domain. Spatio-temporal expression profile showed that except in gonads, the level ofBmFAF was slightly higher in immune tissues than in other tissues, and in molting larvaeBmFAF 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 ofBacillus bombyseptieus orSerratia arcescens, and did not recover within 12 h, whereasBmCecropinA1 was continuously up-regulated and reached the highest degree at 12 h. Over-expression ofBmFAF in BmE cells led to a remarkable decrease of anti-microbial peptides, including BmCecropinA1,BmAttacin andBmMoricin. Moreover, the expression level ofBmFAF could be efficiently knocked down to 50%-60% by dsRNA, at the same time, the expression level ofBmCecropinA1 andBmMoricin was significantly up-regulated. Injection of dsFAF into the 5th-instar day-2 larvae resulted in a decrease ofBmFAF to 45% of the level in larvae injected with dsEGFP at 24 h post injection, and the expression level ofBmCecropinA1,BmAttacin andBmMoricin 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 ofS. 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 thatBmFAFacts as an immune negative regulatory molecule by suppressing the expression of anti-microbial peptides, and down-regulation ofBmFAF resulted in an increase of anti-microbial peptides which would potentially increase the resistance of silkworm larvae to bacterial infection.关键词
家蚕/免疫负调控/BmFAF/表达特征/抗菌肽/存活率Key words
Bombyx mori/immune negative regulation/BmFAF/expression profile/anti-microbial peptide/survival rate引用本文复制引用
马晓娟,李亚明,胡翠美,王菲,夏庆友..家蚕免疫负调控分子BmFAF的功能[J].中国农业科学,2014,(15):3085-3093,9.基金项目
国家“973”计划（2012CB114600）、国家自然科学基金青年科学基金 （）
