家蚕免疫负调控分子BmFAF的功能

doi:10.3864/j.issn.0578-1752.2014.15.018

摘要/Abstract

摘要： 【目的】在细胞和个体水平上探讨家蚕BmFAF的免疫负调控功能，为研究昆虫免疫稳态提供参考。【方法】通过RT-PCR技术克隆BmFAF并进行结构域预测和分析；利用荧光定量PCR检测BmFAF在家蚕各龄期以及5龄第3天幼虫的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；构建BmFAF的细胞表达载体以及合成用于RNAi的dsRNA，通过质粒或dsRNA的转染在BmE细胞中过表达BmFAF或降低BmFAF的表达水平，并利用Western blotting或定量PCR予以确认，同时检测抗菌肽基因表达水平的变化；通过注射dsRNA在个体中降低BmFAF的表达水平，并检测BmFAF对抗菌肽基因表达水平和家蚕感染细菌后存活率的影响。【结果】BmFAF与其他物种FAF家族成员有较高的同源性，与人类HsUBXD8、HsFAF1和果蝇DmCaspar分别具有60%、42%和47%的相似性，并具有保守的UBX结构域。时空表达特征分析表明，除生殖腺外，BmFAF在免疫组织中表达量略高于其他组织，眠蚕期表达量显著高于起蚕期，尤其在2龄和3龄眠蚕中的表达量最高；5龄时BmFAF的表达量普遍较低，而从5龄发育至预蛹期时，BmFAF的表达水平有所上升。免疫诱导表达谱显示，家蚕在注射感染黑胸败血芽孢杆菌或黏质沙雷氏菌0.5 h后，脂肪体中BmFAF的表达水平即显著下降，且在12 h内没有恢复，而抗菌肽BmCecropinA1的表达则持续上升，并在12 h时达到最高。在BmE细胞中过表达BmFAF，抗菌肽BmCecropinA1、BmAttacin和BmMoricin的表达水平与转染对照质粒的细胞中的抗菌肽相比，均显著降低。在细胞中转染dsRNA后，BmFAF的表达水平被有效地降低50%—60%，抗菌肽BmCecropinA1和BmMoricin的表达水平与对照相比均显著升高。对5龄第2天家蚕幼虫注射dsRNA，24 h后通过荧光定量PCR检测到BmFAF的表达水平在注射dsFAF的家蚕中下降至注射dsEGFP对照中的45%；而抗菌肽BmCecropinA1和BmMoricin、BmAttacin的表达水平均被显著上调。这些家蚕通过注射感染黏质沙雷氏菌24 h后，死亡率即低于对照组，在感染72 h后仍有40%左右的家蚕存活，而对照组中则无家蚕存活。【结论】结构分析、表达特征以及细胞和个体水平上的功能研究表明，BmFAF是一个具有免疫负调控作用的分子，通过抑制抗菌肽的表达维持家蚕免疫稳态, 而降低BmFAF的表达水平则导致抗菌肽表达水平升高，从而有助于减缓甚至避免家蚕在感染细菌后的死亡。

关键词: 家蚕 , 免疫负调控 , BmFAF , 表达特征 , 抗菌肽 , 存活率

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

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, 47(15): 3085-3093.

MA Xiao-Juan, LI Ya-Ming, HU Cui-Mei, WANG Fei, XIA Qing-You. Functional Characterization of BmFAF as an Immune Negative Regulatory Molecule in Silkworm (Bombyx mori)[J]. Scientia Agricultura Sinica, 2014, 47(15): 3085-3093.

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