家蚕aly%2fref基因的克隆及其表达蛋白在细胞中的定位.pdfVIP

国蚕学会第七届青年学术研讨会论文集 家蚕aly/ref 基因的克隆及其表达蛋白在细胞中的定位* ＃ ＃ 钟金凤 ，曹广力 ，薛仁宇，贡成良 苏州大学基础医学与生物科学学院，江苏苏州 215 123 摘要：RNA 和输出因子结合蛋白（RNA and export factor binding proteins ，REF ；又称ALY ）参与 RNA 的加工、输出和稳定性。本研究通过RT-PCR 克隆了家蚕aly/ref cDNA 基因（Bmaly/ref ），测 序结果显示该基因的开放读码框为765 bp ，编码254 个氨基酸残基。结构分析显示BmALY/REF 具 有REF 家族的与RNA 结合的结构域RRM ，N 、C 端分别且有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、意大利蜜蜂的ALY/REF 较为接近。将B maly/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 均有分布，但主要分布在细胞核 。研 究结果为进一步探讨BmALY/REF 的功能奠定了基础。 关键词：家蚕；BmALY/REF ；基因克隆；序列分析；细胞定位 图分类号：Q349；S881.2 文献标识码：A 文章编号： Clone and Analysis of Bmaly/ref cDNA Gene and Cellular Localization Analysis of the BmALY/REF ZHONG Jin-Feng, CAO Guang-Li, XUE Ren-Yu, GONG Cheng-Liang (Medical College of Sooch ow University, Suzhou, Jiangsu 215123, China) Abstract: ALY/REF is RNA and export factor binding proteins, which plays important roles of RNA processing、exportation and stability. The aly/ref cDNA gene from Bombyx mori was cloned and sequenced. The whole cDNA sequence covers 765 bp, which contains a complete open reading fragment and encodes a 254 amino acid protein. Structure analysis showed that BmALY/REF contains RNA binding domain (RRM), REF-N and REF-C motif of REF subfamily. Homologous analysis showed that BmALY closed to Ap is mellifera and Tribolium castaneum, and the insects ALY clustered to a group The BmALY gene was inserted into vector pGS21a (+) to prokaryotic expression. The mice were immunized with the Recombinant protein to prepare polyclonal antiserum. Immuno-fluorescence examination show that BmAly protein is both cytoplasmic and nuclear, and mainly located inside nucleus. Above experiment provi