家蚕BmE(spl)-like基因的克隆与表达谱分析

Cloning and Expression Profile Analysis of BmE(spl)-like Gene in Bombyx mori

从本实验室构建的家蚕蛹cDNA文库中筛选到一条新的cDNA序列,生物信息学分析发现其编码的蛋白质序列与果蝇Enhancer of split基因[E(spl)]编码的DNA结合转录调控因子bHLH(helix-loop-helix)有较高的同源性,将该基因命名为BmE(spl)-like(Bombyx mori Enhancer of split like)。该基因的ORF长606 bp,编码201个氨基酸残基,预测蛋白质分子质量22.3 kD,等电点9.7。构建重组表达质粒pET-28a-BmE(spl)-like并在E.coli BL21(DE3)菌株中表达BmE(spl)-like蛋白,经亲和层析和FPLC反向柱纯化融合蛋白His-BmE(spl)-like并制备了抗体效价1∶25 600以上的多克隆抗体。用qRT-PCR方法检测BmE(spl)-like在家蚕成虫发育期的转录水平最高,结合果蝇同源基因的功能,推测该基因与蚕蛾的翅发育相关;该基因在家蚕5龄幼虫各组织中的转录水平以表皮最高,马氏管中最低。Western blotting检测BmE(spl)-like在家蚕5龄幼虫性腺中的表达水平最高,其次是丝腺、表皮、脂肪体、肠组织,在头部、马氏管和气管中的表达水平极低,与qRT-PCR检测的BmE(spl)-like在各组织中的转录水平有些差异。

A novel cDNA sequence was screened from the silkworm pupa cDNA library constructed in our laboratory. Bioinformatics analysis showed that the protein sequence encoded by this gene shares a high homology with the inhibitor of DNA binding bHLH （ basic helix-loop-helix） encoded by Enhancer of split gene [ E（spl） 1 of Drosophila melanogaster, and was thus designated as BmE（spl） -like （ Bombyx mori Enhancer of split like）. It contains an ORF of 606 bp and encodes a protein of 201 amino acids with a predicted molecular mass of 22.3 kD and an isoelectric point of 9.7. We constructed the recombinant expression plasmid pET-28a-BmE（ spl）-Iike and expressed BmE （ spl）-Iike protein in E. coil BL21（DE3）. After purification using affinity chromatography and FPLC reversed-phase column, the expressed fusionprotein His-BmE（spl）-Iike was used to prepare polyclonal antibody whose titer was over 1 ： 25 600. qRT-PCR detec- tion showed that the transcription level of BmE（spl）-like was the highest in adult stage. Based on the functions of homologous genes from D. melanogaster, it is suggested that BmE（spl）-like relates to wing development of Bombyx mori. The transcription level of BmE（spl） -like in vari-ous tissues was the highest in epidermis, and the lowest in Malpighian tubule. Western blotting detection indicated that BmE（spl）-Iike had the highest expression level in gonad, followed by silk gland, epidermis, fat body and intestine, and had the Lowest expression level in head, Malpighian tubule and trachea, showing certain difference with the transcription levels determined by qRT-PCR.