家蚕新基因BmCMC1的表达分析及亚细胞定位研究

cDNA sequence containing a conserved domain CMC was found from the cDNA library constructed by our laboratory and was named as BmCMC1. The BmCMC1 gene contains two exons, and one intron, the ORF is 305 bp and encodes a protein of 118 amino acid residues named as BmCMC1. The predicted protein BmCMC1 has a molecular weight of 13.58 kD and the theoretical pI lies at 8.98. The recombinant fussion protein His-tag BmCMC1 was successfully expressed in E.coli BL21(DE3), and its molecular weight is 17.38 kD. The fussion protein was purified with HiTrap Chelating HP,followed by FPLC, and then analyzed by mass-spectrum. The result of mass-spectrum confirming that the His-tag BmCMC1 was expressed in the right form. The purified fussion protein was used to immunize the male New Zealand rabbit. Then, the antisera (polyclonal antibodies) were harvested. Western Blot analysis revealed that the protein BmCMC1 was expressed in silk glands , ovary of the fifth instar larva, but wasn’t expressed in other tissues. No siganl was detected in the four developmental stages of silkworm. The result of real-time PCR revealed that BmCMC1 mRNA is widespread in the developmental stages of silkworm, and different tissues of the fifth instar larva . According to the immunofluorescence staining, the subcellular localization of the BmCMC1 was analyzed. BmCMC1 accumulated in cytoplasm.These results laid a good foundation for further study of BmCMC1.