关键词: 甜菜夜蛾, 3′RACE-PCR, 泛素延伸基因, 分子进化, 原核表达

Abstract: To identify the nucleotide sequence of ubiquitin cDNA in Spodoptera exigua, a 513 bp cDNA fragment encoding ubiquitin-53aa extension protein from S. exigua lipid was amplified with 3'RACE (rapid amplification of cDNA ends) PCR with primers designed on the N-terminal amino acid sequence of S. frugiperda biquitin extension gene, and the amplified fragment was cloned and sequenced. The ubiquitin-53aa extension gene (ubi-53) in S. exigua was 513 bp in length, includeing 123 bp of 3'untranslated region and 390 bp of encoding region. The encoding region encoded a peptide of 129 amino acid residues, in which there was an ubiquitin fused with a ribosomal L40 protein. A comparison of the deduced amino acid sequence with those of Bombyx mori, S. frugiperda, Drosophila melanogaster, Homo sapienes, SeNPV and BmNPV showed that the amino acid homology rates were 96.9%，98.5%，95.3%，93.0%, 78.8 % and 77.2% respectively. This suggests ube genes in eukaryotes may have a different evolution way from its host viruses. The fragment containing ubi-53 gene was inserted into pET-28a expressive vector, and the expression was induced by IPTG in E. coli BL21(DE3). The fusion protein was identified by Western blot using a mouse monoclonal antibody against bovine ubiquitin.

Key words: Spodoptera exigua, 3′RACE-PCR, ubiquitin-53aa extension protein, molecular evolution, prokaryotic expression