关键词: 薇甘菊, 乙醇酸氧化酶, 基因克隆, 序列分析, 原核表达

Abstract: A full length cDNA encoding a putative glycolate oxidase, designated as MmGO, was identified by using suppression subtractive hybridization (SSH) technique combined with rapid amplification of cDNA ends (RACE) methonds from stems of Mikania micrantha. The MmGO gene was analyzed by bioinformatic tools, then cloned into the expression vector pET-32a(+), and finnally expressed in the prokaryote Escherichia coli Rosetta-gami (DE3). Nucleotide sequence data indicated that the full length cDNA of MmGO gene was 1 363 bp (GenBank accession EU716626), encoding a 369 amino acid protein. MmGO is predicted to encode a 40.32 kDa polypeptide with a isoelectric point of 8.99. Phylogenetic analysis showed that the predicted MmGO was closely related to glycolate oxidase (GO) of Brassica napus. The MmGO gene was then constructed into expression vector pET-32a(+) for expression in prokaryotic cells. The fusion protein (6×His-MmGO) was produced at a high level by prokaryotic expression when induced by 0.1 mmol·L^-1 IPTG at 25℃ for 4 h. The results of Western-blot demonstrated that the fusion protein(6×His-MmGO) could be recognized by anti-6×His monoclonal antibody. The fusion protein molecular mass was about 60 kDa. This size was agreed with the predicted molecular mass. This study established the foundation for future researches on the enzymatic activity and function of the fusion protein (6×His-MmGO).

Key words: Mikania micrantha, glycolate oxidase, gene cloning, sequence analysis, prokaryotic expression