家蚕MYST组蛋白乙酰转移酶基因Bm-mof的克隆表达和转录活性检测

Cloning, expression and transcriptional activity of Bm-mof, an MYST histone acetyltransferase gene in the silkworm, Bombyx mori L.

MYST组蛋白乙酰转移酶(histone acetyltransferase,HAT)广泛存在于从酵母到人的真核生物中,在真核生物的转录调控中起着重要的作用。利用NCBI已登录的其他物种该基因的氨基酸序列与家蚕的基因组框架图和表达序列标签(expres sedsequence tags,EST)数据库进行电子克隆,获得了家蚕中的同源基因。该基因长1575bp(GenBank登录号为DQ442997),开放阅读框(ORF)长1326bp,无内含子。基因编码442个氨基酸,预测蛋白质的分子量为51.4kD。序列中有HAT核心结构域、锌指结构域和染色质域3个保守的结构域,与其他物种同源基因具有较高的序列相似性。RT-PCR结果表明该基因在本实验所检测的家蚕各时期和组织中均有表达。将该基因用亚克隆的方法导入到pET50b载体中并成功地进行了原核表达,表达出了带有6个组氨酸和1个Nus.Tag标签的重组蛋白。

MYST acetyltransferase widely exists in the eukaryon from yeast to human and it is now known to play a major role in the regulation of eukaryotic transcription. Based on the reported amino acid sequences of other organisms and the scaffold and EST sequences of the silkworm Bombyx mori L., the MYST acetyltransferase gene of the silkworm was obtained with silico cloning. The gene is 1 575 bp in length （GenBank accession number： DQ442997）, containing an ORF of 1 326 bp and no intron. The predicted protein consists of 442 amino acids with the molecular weight of 51.4 kD. It contains three conservative domains, i.e., MYST core domain, zinc finger and chromodomain, and has high similarity with homologous genes in other species. The RT-PCR experiment indicated that the gene was expressed in all tested tissues and stages of the silkworm. Prokaryotic expression of the gene reeornbinant with six-His tag and a Nus· Tag was successfully carried out through sub-cloning into pET50b vector.