关键词: 家蚕, BmAly/REF, 基因克隆, 序列分析, 细胞定位, 基因芯片

Abstract: RNA and export factor binding proteins (Aly/REF) play important roles in RNA stability, processing and nuclear export. Referring to the sequence of Bombyx mori aly gene (GenBank accession no.DQ497195.1), the aly/ref gene (Bmaly/ref） from the silkworm, B. mori, was cloned via RT-PCR. The sequencing result showed that the open reading frame(ORF) of Bmaly/ref is 765 bp in length, encoding 254 amino acid residues, and the amino acid sequence of BmAly/REF showed 49.7% and 52.7% identities with the homologs of Drosophila melanogaster and Mus musculus, respectively. The structure prediction displayed that BmAly/REF contains RNA binding domain (RRM), REF-N and REF-C motif of REF subfamily. Phylogenetic analysis showed that the insect Aly/REF proteins clustered to a group and the BmAly/REF was close to Aly/REF proteins of Apis mellifera and Tribolium castaneum. The BmAly gene was inserted into expression vector pGS21a (+), and then the recombinant protein was expressed in Escherichia coli and used to immunize mice to prepare the antibody against BmAly/REF. Immuno-fluorescence examination showed that BmAly/REF was distributed in both cytoplasm and nucleus of the cell, but mainly located in the nucleus. Microarray data analysis showed that Bmaly/ref gene was highly expressed in tissues of the day- 3 5th instar larvae of the silkworm. The results suggest that BmAly/REF is likely to play important roles in RNA nuclear export, which lays a good foundation to further investigate the function of BmAly/REF.

Key words: Bombyx mori, BmAly/REF, gene cloning, sequence analysis, cellular localization, microarray