条斑紫菜泛素结合酶基因的cDNA序列克隆与分析

泛素蛋白酶体系统（ubiquitinproteasomes system，UPS）是真核生物重要的翻译后修饰系统，参与了许多重要的生理反应，同时也参与了蛋白质的质量控制和细胞的稳态维持，因此研究条斑紫菜UPS有助于阐明条斑紫菜在逆境胁迫下通过UPS降解异常蛋白和调节蛋白的生物学活性。泛素结合酶（ubiquitinconjugating enzyme，E2）是UPS的重要组成部分。根据电子克隆结果指导RT-PCR实验，成功克隆了条斑紫菜泛素结合酶基因（PyE2）的cDNA序列（GenBank accession：FJ232910）。该cDNA序列含有长444 nt的完整ORF，编码蛋白（PyE2）的分子量16.6 ku，长147 AA。PyE2与其它物种的E2具有高度的一致性和相似性，反映出E2在进化上相当保守。PyE2含有E2活性位点的保守序列及半胱氨酸残基，其三维结构与人的I类E2高度一致。PyE2不含C端和N端延伸结构，所以在条斑紫菜E2家族中属于I类E2。

cDNA cloning and characterization of a novel ubiquitin conjugating enzyme gene from Porphyra yezoensis Ueda

The ubiquitin proteasome system (UPS) plays an important role in post translational process, which is responsible for a diverse array of biologically important cellular processes. The UPS is also involved in the protein quality control, which maintains the health of the cell. Thus, the UPS provides a clue for understanding of the molecular mechanisms underlying various tolerances of organism to diversiform adverse conditions as well as stress combination. Ubiquitin conjugating enzyme (E2) is a critical component of UPS. Cloning of ubiquitin conjugating enzyme gene (PyE2) of Porphyra yezoensis Ueda will facilitate us to investigate tolerance of P. yezoensis to diversiform adverse conditions and degradation of enable protein by UPS. cDNA sequence (GenBank accession: FJ232910) of PyE2 was isolated successfully through experiment of RT-PCR under direction of in silico cloning. This cDNA contained a 444 nt of complete open reading frame (ORF) coding a 16.6 ku protein (PyE2) of 147 amino acids sequence. PyE2 had significant amino acid sequence identity and similarity with E2s from other organism, which indicated E2 was highly conserved in the evolution. PyE2 contained conserved sequence and cysteine residue of active site of E2 family, and tertiary structure of PyE2 was similar to human class I E2. PyE2 was a class I member of E2 family of P. yezoensis according to bioinformatics analysis.