| Post-translational modification of protein is an important regulatory mechanism for protein function,including phosphorylation,acetylation,glycosylation,ubiquitination,and ubiquitination-like modifers,and so on.Neddylation is a kind of protein modification mechanism similar to ubiquitination,which is involved in important biological processes such as cell proliferation and development,DNA damage repair,protein stability,transcriptional regulation,chromosome formation,apoptosis and autophagy.And it is also associated with the replication of some virus.Silkworm is an important economic insect in production,and it is also an important model organism in the field of scientific research.Bombyx mori nuclear polyhedrosis virus(Bm NPV)is an important pathogen of silkworm,which has caused heavy losses to sericulture production every year.In this study,the characteristics of NEDD8 gene were studied;a key gene of Neddylation,and the relationship between Neddylation and Bm NPV replication was discussed.The results are as follows:(1)Based on NCBI database,the NEDD8 gene was successfully cloned from silkworm.The gene has an ORF with 234 bp encoding 77 amino acids with a molecular weight of about 8.47 k Da and no signal peptide was found.The amino acid sequence of NEDD8 was highly conserved with other model organisms and the ubiquitin-related proteins of silkworm,and the homozygous amino acid sequence between NEDD8 and ubiquitin in silkworm was the highest.QRT-PCR results showed that NEDD8 was expressed in different tissues of the fifth instar larvae of silkworm,with the highest expression in the head,followed by the silk gland,and the lowest in the testis and ovary.The expression level of NEDD8 m RNA was increased from the 3rd day of the fifth instar larvae to the third day after pupation,and reached the highest at the 9th day after pupation.Then it decreased in moth.The recombinant Bm NPV-GFP-NEDD8 was transfected into Bm N cells.It showed that NEDD8 was distributed throughout the Bm N cells,and aggregated as dots in cytoplasms at 48 h after infection.(2)The effects of the specific inhibitor of Neddylation MLN4924 on Bm NPV were evaluated.The results showed that the MLN4924 significantly inhibited the BV production and polyhedron formation.The viral DNA replication and viral protein expression were inhibited,while the BV virus particles' adsorption and invasion to the Bm N cells were not affected.(3)Immunoprecipitation and LC-ESI-MS / MS mass spectrometry were used to screen NEDD8 modified proteins in silkworm cells.A total of 28 possible NEDD8 modified substrate proteins were identified from Bm N cells,and 17 of them could be matched to GO terms.Among them,42.86% and 46.43% of the protein were involved in binding and catalytic activity under the molecular function,while 26.83%,26.83% and 19.5% were classified into cells,cell parts and organelles respectively under the cellular component.Under the biological process,30%,24% and 8% proteins were involved in the metabolic process,cellular process and single-organism process.KEGG pathway analysis showed that NEDD8-related proteins were involved in 33 channels such as Ubiquitin mediated proteolysis,Proteasome and m RNA surveillance pathway,indicating that NEDD8 may be involved in the process of apoptosis,protein degradation and transcription regulation. |
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