Molecular characterization and immunohistochemical localization of a novel troponin C during silkworm development.

We have cloned and sequenced a novel Bombyx mori gene that encodes a protein having a high degree of homology with other known troponin C (TnC) proteins. The amino acid sequence, DX[DN]X[DSG]X(6)E, a highly conserved putative Ca(2+) -binding motif found in loops within the globular domains of previously identified TnC proteins, is also present in BmTnC. We have expressed and purified to homogeneity a His-tagged BmTnC fusion protein having a molecular weight of approximately 21.6 kDa. We have used this purified fusion protein to produce polyclonal antibodies against BmTnC for Western blot analyses. These analyses have revealed that BmTnC is expressed in the larval head, the Malpighian tubule, the epidermis, the testis, and the gut, as has been confirmed by immunohistochemistry. In addition, real-time reverse transcription/polymerase chain reaction has shown that BmTnC mRNA levels differ substantially among these tissues. Our findings indicate that BmTnC is selectively expressed in the muscular tissues of the silkworm, including portions of the head, the Malpighian tubule, the body wall, and the gut.