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以猕猴桃属植物中果实维生素C含量最高的阔叶猕猴桃(Actinidia latifolia Merr.)果实为试材,经RT-PCR扩增获得约1.0kb的L-半乳糖脱氢酶cDNA片段。生物信息学分析表明,该cDNA片段长为997bp,最大开放阅读框为960bp,可编码319个氨基酸残基,命名为AlGalDH,GenBank登录号为EU525846,核苷酸序列及其推导的氨基酸序列与已知其它植物GalDH核苷酸、氨基酸序列间的同源性分别在76%和70%以上,且具有醛酮还原酶的保守结构域。构建了其原核表达载体pET-AlGalDH并转化大肠杆菌BL21,经0.1mmol.L-1IPTG诱导,获得具有较高活性的表达融合蛋白6×His-AlGalDH。经Ni-His亲和磁珠分离纯化,获得单一的融合目的蛋白条带,测定其酶活为120pmol.min-1.mg-1。
Actinidia latifolia Merr., A fruit of Actinidia with the highest content of vitamin C in fruit of Actinidia, was amplified by RT-PCR to obtain a cDNA fragment of L-galactose dehydrogenase of about 1.0 kb. Bioinformatics analysis showed that the cDNA fragment was 997bp in length and 960bp in open reading frame. It encoded 319 amino acid residues, named AlGalDH, and its GenBank accession number was EU525846. The nucleotide sequence and its deduced amino acid sequence were Known to other plants GalDH nucleotide, amino acid sequence homology between 76% and 70%, respectively, and has a conserved domain of aldo-keto reductase. The prokaryotic expression vector pET-AlGalDH was constructed and transformed into E.coli BL21. After induced by 0.1 mmol.L-1 IPTG, the expressed fusion protein 6 × His-AlGalDH with high activity was obtained. Purified by Ni-His affinity magnetic beads, to obtain a single fusion protein bands, the enzyme activity was measured as 120pmol.min-1.mg-1.