阔叶猕猴桃果实GalDH cDNA克隆及其在大肠杆菌中的表达  

Cloning of L-galactose Dehyrogenase Gene from Actinidia latifolia Merr. and Its Expression in E.coli

作　　者：尚增振[1] 王小华[1] 马锋旺[1] 梁东[1] 

机构地区：[1]西北农林科技大学园艺学院,陕西杨凌712100

出　　处：《园艺学报》2009年第12期1741-1748,共8页Acta Horticulturae Sinica

基　　金：国家自然科学基金项目(30871700);西北农林科技大学拔尖人才支持计划项目

摘　　要：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。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 AlGalDH （ GenBank accession No. EU525846） was amplified by reverse transcription polymerase chain reaction （RT-PCR） and then cloned. Bioinformatics analysis indicated that the length of eDNA was 997 bp, which contained an open reading frame of 960 bp and encoded a protein of 319 amino acid residues. The homology analysis demonstrated that AlGalDH shared high similarities of nucleotides and deduced amino acids with those in other plants over 76% and 70% respectively. The forms of AIGalDH included one Aldo-keto reductases conversed domains. The AIGalDH was then successfully subcloned into pET-32a （ ＋ ） to construct its prokaryotic expression vector pET-AlGalDH. After transformation into E. coil BL21, the fusion protein （6 × His-AlGalDH） was produced at a high level induced by 0. 1 mmol ·L ^-1 IPTG. One single band of protein with the His-tag was purified by Ni- His Bind Resin and then used as the template to analyze the enzyme activity which showed 120 pmol ·min^-1 · mg^-1.

关 键 词：阔叶猕猴桃 GalDH 克隆 原核表达 

分 类 号：S663.4[农业科学—果树学] Q78[农业科学—园艺学]