角毛壳菌泛素结合酶基因的克隆和特性分析

Cloning and Characterization Analysis of Ubiquitin Conjugating Enzyme Gene fromChaetomium cupreum

利用BlastX将角毛壳菌(Chaetomium cupreum)几丁质诱导的菌丝体cDNA文库中的ESTs序列与NCBI的Nr(Non-redundant)数据库进行相似性搜索,获得泛素结合酶(ubiquitin conjugating enzyme,E2)基因的ESTs.以此结果设计引物进行RT-PCR,克隆该基因的cDNA序列(CcE2).该序列长600bp,开放阅读框444bp,编码147个氨基酸组成的多肽,蛋白分子量为16.6ku,是亲水性蛋白.BlastP分析表明该基因进化相当保守,与其他真菌的E2具有高度的相似性;三维结构分析显示CcE2仅包含一个功能结构域,在结构域的中央是决定CcE2活性的半胱氨酸残基活性位点.

ESTs of ubiquitin conjugating enzyme was obtained from mycelia cDNA library of Chaetomium cupreum by inducement of chitin through BlastX search with Non-redundant database of NCBI. CcE2 was cloned by RT-PCR. CcE2 was hydrophilic protein with full length of 600 bp with an open read frame of 444 bp and encoded 147 amino acids with 16.6 ku protein molecular weight. The BlastP results showed that CcE2 had significant amino acid sequence similarity with E2s from other fungi. Tertiary structure of CcE2 contained a functional structural domain. An active site of cysteine residue was located in the middle of functional structural domain.