家蚕BmCaspase-8-Like(BmCasp8L)的免疫负调控功能

Functional Characterization of BmCaspase-8-Like(BmCasp8L) as an Immune Negative Regulatory Molecule in Silkworm(Bombyx mori)

【目的】在个体和细胞水平上探讨家蚕(Bombyx mori)BmCaspase-8-Like(BmCasp8L)的免疫负调控功能,为研究昆虫免疫负调控机制提供参考。【方法】通过RT-PCR技术克隆BmCasp8L并进行结构域预测和进化分析;利用荧光定量PCR检测BmCasp8L在家蚕各龄期、5龄第3天及预蛹期各组织中的时空表达特征和家蚕感染细菌后的免疫诱导表达特征;合成用于RNAi的dsRNA,通过注射dsRNA在个体中降低BmCasp8L的表达水平,并检测对抗菌肽基因表达水平的影响;构建BmCasp8L的细胞表达载体,通过质粒或dsRNA的转染在BmE细胞中过表达BmCasp8L或降低BmCasp8L的表达水平,并利用Western blot或定量PCR予以确认,同时检测抗菌肽基因表达水平的变化及转录因子BmRelish的切割。【结果】BmCasp8L与鳞翅目Caspase-6、哺乳动物Caspase-8同源,其序列与BmDredd、DmDredd N端分别具有61%、42%的相似性,但缺少C端的Caspase结构域。时空表达特征分析表明,BmCasp8L在眠蚕期表达量高于起蚕期,在预蛹期、蛹第7天、蛾第1天表达量明显升高;在5龄第3天幼虫体内,BmCasp8L在血细胞中的表达量明显高于其他组织,而在预蛹期,BmCasp8L主要在丝腺中表达。免疫诱导表达谱显示,5龄第3天家蚕在注射感染黑胸败血芽孢杆菌或粘质沙雷氏菌1 h内,BmCasp8L的表达水平上升,此后逐渐恢复正常。对5龄第2天家蚕注射dsCasp8L或dsEGFP,24 h后通过荧光定量PCR检测到BmCasp8L的表达量在注射dsCasp8L的家蚕中显著下调,而抗菌肽BmCecropinA1的表达水平显著上调。在BmE细胞中过表达BmCasp8L,抗菌肽BmCecropinA1的表达水平与对照相比显著降低,且转录因子BmRelish的切割受到抑制。在细胞中转染dsRNA后,BmCasp8L的表达水平被有效降低,抗菌肽BmCecropinA1的表达水平显著上调。【结论】进化分析、表达特征以及细胞和个体上的功能研究表明BmCasp8L是一个具有免疫负调控作用的分子,通过抑制BmRelish的切割,抑制抗菌肽的表达,从而负调控Imd信号通路,降低BmCasp8L的表达水平则导致抗菌肽表达水平升高,有助于家蚕抵御微生物病原的侵染。

【Objective】The objective of this study is to characterize BmCaspase-8-like（BmCasp8 L） 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 Bm Casp8 L by RT-PCR. Then fluorescence quantitative PCR was used to investigate the spatial-temporal expression profile of Bm Casp8 L at different development stages and in different tissues extracted from the 3 rd day of 5 th-instar larvae and prepupa, as well as the larvae body after bacterial infection. The dsRNA for RNAi was synthesized to silence Bm Casp8 L in the B. mori larvae, and the effect of Bm Casp8 L on expression of the anti-microbial peptides was studied. The plasmid for expressing Bm Casp8 L in cells was constructed. After transfection of BmE cells with the expression constructs or ds RNA, Western blot or quantitative PCR was performed to confirm the over-expression or knock-down of Bm Casp8 L in cells. Meanwhile, the change in expression of the anti-microbial peptides and cleavage of nuclear transcription factor BmRelish were detected.【Result】Bm Casp8 L is homologous to Lepidoptera Caspase-6 and mammalian Caspase-8. The similarity between BmCasp8 L 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 Bm Casp8 L level was higher than in newly exuviated ones, and the Bm Casp8 L expression level was significantly increased in the prepupa, 7 th day of pupa and 1 st day of moth stage. The Bm Casp8 L expression level in the hemocyte was significantly higher than in other tissues in the 3 rd day of 5 th-instar, but in the prepupa stage, it was mainly expressed in the silk gland. The Bm Casp8 L expression level in the 3 rd day of 5 th-instar larvae increased within 1 h post infection of Bacillus bombyseptieus or Serratia marcescens, and then gradually returned to normal. Injection of dsCasp8 L or dsEGFP into the 2 nd day of 5 th-instar larvae, the expression of Bm Casp8 L was significantly down-regulated in B. mori injected with dsCasp8 L, while the expression of antimicrobial peptide BmCecropinA1 was up-regulated after 24 h. Over-expression of Bm Casp8 L in BmE cells led to a remarkable decrease of the anti-microbial peptide BmCecropinA1. In addition, over-expression of Bm Casp8 L suppressed the cleavage of the transcription factor BmRelish. Moreover, the expression level of Bm Casp8 L 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 BmCasp8 L 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 Bm Casp8 L resulted in an increase of anti-microbial peptides which would potentially increase the resistance of B. mori larvae to bacterial infection.