A Novel Digestive Protease Chymotrypsin-Like Serine Contributes to Anti-BmNPV Activity in Silkworm (Bombyx Mori).

Abstract

Serine proteases (SPs) are important proteases in the digestive system of lepidopteran insects. They play important roles in protein digestion, coagulation, signal transduction, hormone activation, inflammation and development. Blood-borne pyosis caused by Bombyx mori nuclear polyhedrosis virus (BmNPV) has caused serious harm to sericulture. At present, the scientific problems of BmNPV infection and silkworm resistance to BmNPV infection have been the focus of many scientists, but the molecular mechanism needs further research and exploration. Based on the results of label-free quantitative protein proteomics of the midgut digestive juice of different resistant strains in our laboratory, we successfully screened a differentially expressed candidate protein (DEP), B. mori chymotrypsin-like serine protease (BmCLSP), and comprehensively analyzed the biological characteristics and anti-BmNPV function of BmCLSP. The open reading frame (ORF) of BmCLSP is 891 bp, encoding 296 amino acid residues. The analysis of the domain structure showed that there was a signal peptide and a trypsin-like serine protease domain, Tryp_SPC, in the BmCLSP protein. Semi-quantitative and real-time fluorescence quantitative PCR analysis showed that the BmCLSP gene was highly expressed in the fifth instar larvae of silkworm, and specifically expressed in the midgut. The expression level of BmCLSP in the BmNPV resistant strain A35 was higher than that in the sensitive strain P50. Virus amplification analysis showed that the relative expression level of VP39 was significantly lower than that of the control group after infection of silkworm larvae and BmN cells with BmNPV treated with recombinant BmCLSP at an appropriate concentration. Furthermore, our overexpression of BmCLSP in BmN cells significantly inhibited the expansion of BmNPV. In summary, the results of this study indicate that BmCLSP has anti-BmNPV activity in silkworm, and can significantly inhibit the proliferation of BmNPV in silkworm. It offers a promising avenue for silkworm anti-virus breeding.