Cloning, Expression And Acetylation Studies On BmNAT Gene In Silkworm, Bombyx Mori

N-acetyltransferase?NAT?is a kind of enzyme that catalyzes the transfer of acetyl group between acetyl-co A and amine.The NAT in insect is mainly involved in the pigmentation,control of circadian rhythm,the biosynthesis of chitin,pesticides design and so on.Bombyx mori is a typical model leppidoptera insect,and it also can be used as bioreactor for production of recombinant protein.At present,the related reports about NAT in Bombyx mori are rare.The regulation mechanism about gene BmNAT has not been reported.According to the released Bombyx mori NAT gene in GenBank,we firstly carried out the bioinformatics analyses.BmNAT?Bombyx mori NAT,BmNAT?gene is 1690 bp in length that contains a 1065 bp ORF,encoding a protein of 355 amino acid residues with a predicted molecular weight of 39.894 kDa?GenBank accession number: XP₀04932777.1?and isoelectric point is 5.609.Homologous analysis by multi-alignment suggested that BmNAT showed the highest and lowest homology with its homologs from BmNAT?XP₀04932776.1?and Papilio polytes,respectively.BmNAT was cloned into the prokaryotic expression vector pET-28a?+?and the recombinant plasmid was transformed into E.coli BL21?DE3?.After being induced with IPTG,this gene was successfully expressed.The molecular weight of this fusion protein was about 43 kDa by SDS-PAGE?His tag was weight of 3.56 kDa,and BmNAT was weight of 39.894 kDa?,after purified to immunize New Zealand rabbit for the preparation of polyclonal antibody.The titer of the polyclonal antibody reaches 1:12800 measured by ELISA,and the specificity of the antibody was well using Western blotting.Total RNA and protein of different developmental stages and different tissues of the 5th instar larvae were extracted to explore the transcriptional and protein levels of BmNAT presented diverse patterns as shown by qRT-PCR and Western blotting.BmNAT expression levels are highest in head,moth and egg stages,concluding that BmNAT might play a role in these tissues or developmental stages.Subcellular Localization displayed that BmNAT was distributed in the cytoplasm and nucleus.Overexpression of BmNAT in BmN cells was performed to clarify its functions though transfecting recombinant plasmid p IEx-1-BmNAT into BmN cells.Then Flow Cytometry indicated that BmNAT may have influence on the cell cycle.The preious work sgguested that BmNAT contains an acetylated site.Using site-specific mutagenesis,immunoprecipitation and western blotting,we further identified the acetylation of BmNAT itself,which lays a foundation for understanding the new gene regulatory mechanism at the post-translational modification level in the silkworm,Bombyx mori.