Functional analysis of the RNF181 gene reveals its role in the innate immune response of silkworms, Bombyx mori.

Abstract

The RING finger E3 ligase group is a broadly distributed subfamily of E3 ubiquitin ligases characterized by the presence of a RING domain and is involved in various immunological responses. In this study, Ring finger protein 181 (RNF181) in Bombyx mori (B. mori) was identified and investigated its immunoregulatory function. The BmRNF181 gene is located on chromosome 6 and contains an open reading frame of 441 base pairs, encoding a protein consisting of 146 amino acids. The predicted molecular weight is 16.71 kDa, with a the theoretical isoelectric point of 6.50. The encoded protein possesses a characteristic RING domain and seven predicted phosphorylation sites. The recombinant protein was successfully expressed and purified using the pET-28a-BmRNF181 construct. Quantitative real-time PCR (qRT-PCR) analysis revealed that BmRNF181 was expressed in all examined tissues, with the highest level in the fat body and the lowest in the Malpighian tubules. Developmental expression profiling showed that the highest expression level occurs at the fifth instar larval stage. Following challenge with four different pathogens (Escherichia coli (E. coli), Beauveria bassiana (B. bassiana), Bacillus thuringiensis (B. thuringiensis), and B. mori nuclear polyhedrosis virus (BmNPV)). BmRNF181 expression in the fat body was significantly upregulated. Moreover, RNA interference-mediated knockdown of BmRNF181 dramatically increased the expression of antimicrobial peptide genes. These findings suggest that BmRNF181 acts as a negative regulator of the silkworm immune response.