关键词: 家蚕, 泛素化, 类泛素样蛋白, NEDD8, 表达分析, 细胞定位

Abstract: 【Aim】 NEDD8 (neural precursor cell expressed, developmentally down-regulated 8) is an important post-translational modification protein, and plays regulatory roles in the function of its substrate proteins. This study aims to explore the function of NEDD8 in the silkworm,  Bombyx mori. 【Methods】 RT-PCR technology was used to clone the full-length ORF of  NEDD8  from BmN cells. The expression of NEDD8  was detected by real-time quantitative PCR (qRT-PCR) in different developmental stages, different tissues of the 5th instar day-3 larvae, and BmNPV infected BmN cells of B. mori. eGFP-fused NEDD8 was expressed in BmN cells by Bac to Bac system and observed under confocal microscope. Western blotting with GFP antibody was performed to verify the expression of GFP-NEDD8 fusion protein in BmN cells. 【Results】 NEDD8 was cloned. Sequence analysis showed that NEDD8 is highly conserved, and has the highest amino acid sequence identity with silkworm ubiquitin. Real-time quantitative PCR analysis showed that NEDD8 was differentially expressed in various tissues of silkworm with the highest expression level in the head, and then in silk gland, and the lowest expression level in testis and ovary. During the pupal stage, the expression level of NEDD8 gradually increased from the 5th instar day-3 larvae to 3-day-old pupae, while significantly decreased in adults. When BmN cells were challenged with BmNPV, the expression level of NEDD8 increased at the early and very late stages of BmNPV infection. GFP-NEDD8 fusion expression analysis showed that NEDD8 was distributed throughout the BmN cells. At 48 h post infection, aggregations of NEDD8 were formed in the cytoplasm of BmN cells. 【Conclusion】 The NEDD8 sequence is highly conserved among different species. It has the highest expression level in the head of  B. mori larva, and increases gradually during the pupal stage of the moth. NEDD8 is localized in the whole BmN cells and may be involved in BmNPV replication. The results lay the foundation for further study of the biological function of NEDD8 and the modification mechanism of its substrate proteins in B. mori.

Key words: Bombyx mori, ubiquitination, ubiquitin-like protein, NEDD8, expression analysis, cellular localization