家蚕新基因BmY14的表达分析和亚细胞定位研究

Y14 is a kind of RNA-binding protein which plays a major role in RNA surveillance, another name for NMD (nonsense mediated mRNA decay). According to the large-scale scanning of the cDNA library of pupae phase of silkworm, a 501 bp cDNA was identified which encoded a protein had high sequence homology with a known protein Y14, possessing a conservative RNA binding motif. We named this gene BmY14 (Bombyx mori Y14). This gene has an ORF encoding 166 amino acid residues with the predicted molecular weight of 18.4 kDa. The ORF of the gene was cloned into the prokaryotic expression vector and the recombinant plasmid was transformed into E.coli BL21(DE3). After being induced with IPTG, this gene was successfully expressed. Mass spectrogram was carried out to make sure that it was the interested protein. After being purified with Magnet HisTM Ni-Particles, the recombinant protein was immunized as an antigen against a rabbit. ELISA and Western blot analysis revealed that the antibody specificity is high. It was also detected with ELISA and Western blot that the protein was expressed in several silkworm tissues and 4 developmental phases, with the highest expression in pupa in periods, and BmY14 express most in aerocyst, then blood and organs of generation in tissues. Using fluorescent antibody for subcellular localization associated with DAPI counterstain in Bm5 cell, it was found that BmY14 was preliminarily localized in the nucleus. It was confirmed that BmY14 existed mainly in nuclear, and it was proposed that it might play an important role in the silkworm metamorphosis development, particularly, in the maturity of genital and aerocyst.