The expression analysis of cysteine proteinase-like protein in wild-type and nm2 mutant silkworm (Lepidoptera: Bombyx mori)

• The mutant of non-molting in the 2nd instar (nm2) is a recently discovered mutant of Bombyx mori.
• In the mutant, the transcriptional and translational expression level of BmCP-like was higher than that of the wild-type.
• The BmCP-like play a key role in the degradation of the cuticle, and might participate in the immune systems of silkworm.

The mutant of non-molting in the 2nd instar (nm2) is a recently discovered mutant of Bombyx mori. The mutant cannot molt and exuviate and died successively in premolting of 2nd instar. In this study, two dimensional gel electrophoresis (2-DE) was performed to screen the differential expression of epidermis proteins in pre-molting larvae of 2nd instar between the wild-type and nm2 mutant. Interestingly, a cysteine proteinase-like (BmCP-like) protein in nm2 was significantly higher than that of the wild-type. The transcription profiles of BmCP-like gene were investigated by quantitative real-time PCR (qRT-PCR), and the result revealed that BmCP-like mRNA was remarkably higher in nm2 than that of the wild-type. The transcription level of BmCP-like was high in the epidermis while low in the midgut and hemocytes, and fluctuate with development, while the highest in the newly molted larvae of 3rd and lowest in the pre-molting of the 1st and 2nd instar. The body of injected BmCP-like RNAi of 2nd larvae formed a dark spots around the injection place. These results suggested the BmCP-like gene play a key role in the degradation of the cuticle and epidermis layer during molting of 1st and 2nd instar silkworm. Furthermore, the ORF of BmCP-like gene in nm2 was the same to the wild-type. These studies give us a hint that BmCP-like gene maybe not the major gene responsible for nm2, but BmCP-like gene might participate in the immune systems of silkworm, and the upregulation of BmCP-like transcription in the nm2 mutant might be induced by the disadvantages that limit the growth and development of silkworm in order to survive.