The Silkworm Bmycacr Prokaryotic Expression And Subcellular Localization

YcaCR protein contains a conserved ycaC-related domain, belonging to isochorimatase superfamily. It is predicted that the function of ycaC-related protein is similar to isochorimatase, but the specific substrate and biological function is unclear.A unique new Bombyx mori cDNA was identified from the cDNA library constructed by our laboratory after Blast based on NCBI database. The gene was named BmycaCR (Bombyx mori BmycaCR) and the accession number of BmycaCR is GU292794. It encodes a protein containing a conserved ycaC-related domain. The predicted molecular weight is approximately 22.838 kD. We cloned the ORF of this gene into the prokaryotic expression vector pET-28a(+) and expressed it in E. coli Rosetta (DE3). After induced with IPTG, the recombinant protein was successfully expressed. After being purified with Magnet His^TM Ni-Particles, the fusion protein was used to immunize a male New Zealand rabbit. Then, polyclonal antibody was harvested; the titer of the polyclonal antibody reached 1:6400 measured by ELISA. We used real-time fluorescent quantitative PCR and Western blot to compare the transcriptional and expression level of BmycaCR mRNA in different tissues of the fifth instar larva and different stages of silkworm. The results showed that the transcriptional and expression level of BmycaCR gene in four stages of silkworm was highest in the egg, and lowest in the moth. In fifth instar larva, the transcriptional level of BmycaCR gene was found in different larva's tissues following from high to low: testis, trachea, fat body, ovary, epidermis, gut, blood, Malpighian tubule, silk glands and head. Western blot analysis revealed that the BmycaCR was expressed in fat body, trachea, epidermis, gut, ovary and Malpighian tubule, but not detected in head, blood and silk glands. Immunocytochemistry in BmN cells showed that BmycaCR existed in the nucleus and cytoplasm, and most presented in the region near the cell membrane. It laid a good foundation for further studies on the biological function of BmycaCR gene.