关键词: 阔叶猕猴桃, GalDH, 克隆, 原核表达

Abstract: Abstract: Using fruits of Actinidia latifolia Merr. with the highest content of vitamin C in Actinidia as materials, the fragment of L-galactose dehydrogenase (GalDH) gene designated A lGalDH (GenBank accession No. EU525846 ) was amp lified by reverse transcrip tion polymerase chain reaction ( RT-PCR ) and then cloned. Bioinformatics analysis indicated that the length of cDNA was 997 bp, which contained an open reading frame of 960 bp and encoded a p rotein of 319 amino acid residues. The homology analysis demonstrated that A lGa lDH shared high similarities of nucleotides and deduced amino acids with those in other plants over 76% and 70% respectively. The forms of A lGalDH included one Aldo2keto reductases conversed domains.
The A lGa lDH was then successfully subcloned into pET232a ( + ) to construct its p rokaryotic exp ression vector pET2A lGalDH. After transformation into E1coli BL21, the fusion p rotein (6 ×His2A lGalDH) was produced at a high level induced by 011 mmol·L - 1 IPTG. One single band of p rotein with the His-tagwas purified by Ni-His Bind Resin and then used as the temp late to analyze the enzyme activity which showed 120 pmol·min-1 ·mg-1.

Key words: Actinidia latifoliaMerr. , L-galactose dehydrogenase, GalDH, cloning, prokaryotic expression