家蚕Aly/REF的基因克隆、序列分析及其细胞定位

Cloning, sequence analysis and cellular localization of Aly/REF from the silkworm, Bombyx mori

RNA和输出因子结合蛋白(RNA and export factor binding proteins,REF/Aly)参与RNA的稳定、加工和输出。本研究参照已公开的家蚕Bombyx mori aly序列(GenBank登录号:DQ497195.1),通过RT-PCR克隆了家蚕aly/ref基因(Bmaly/ref)。测序结果显示,该基因的开放读码框为765bp,编码254个氨基酸残基,BmAly/REF与黑腹果蝇Drosophila melanogaster、小鼠Mus musculus的同源体的氨基酸序列一致性分别为49.7%和52.7%。结构预测结果显示,BmAly/REF具有REF家族的与RNA结合的结构域RRM,N和C端分别具有REF-N和REF-C基序。进化分析结果显示,昆虫的ALY/REF聚为一类,BmAly/REF与赤拟谷盗Tribolium castaneum、意大利蜜蜂Apismellifera的Aly/REF较为接近。将Bmaly/ref基因克隆进pGS21a(+)载体进行原核表达,重组蛋白免疫小鼠,获得鼠抗BmAly/REF多抗。免疫荧光实验结果显示,BmAly/REF在细胞质和细胞核中均有分布,但主要分布在细胞核中。芯片数据分析显示Bmaly/ref基因在家蚕幼虫5龄第3天各组织中均有较高水平的表达。研究结果显示BmAly/REF可能在RNA的核输出方面发挥作用,为进一步探讨BmAly/REF的功能奠定了基础。

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.