中文摘要：

通过RT-PCR从美味猕猴桃（Actinidia deliciosa）果实中克隆了D-半乳糖醛酸还原酶（GalUR）全长cDNA序列,并分析其序列特征,进行原核表达,制备特异抗体,分析其mRNA及蛋白表达水平与果实发育过程及不同组织中抗坏血酸（AsA）合成和积累的关系。结果表明：克隆的GalURcDNA（GenBank登录号为GU339035）包含的最大开放阅读框（open reading frame,ORF）为990bp,编码329个氨基酸残基,预测分子量为37kD,与报道的草莓等其他植物GalUR具有较高的相似性。构建的pET-32a（＋）-GalUR载体在大肠杆菌BL21（E.coliBL21）中异源表达后,获得了主要以包涵体存在约58kD的融合蛋白GalUR-His（His约21kD）。以该蛋白制备抗体,与重组蛋白的Western杂交表明该抗体能与GalUR蛋白发生特异反应。对猕猴桃可溶性蛋白杂交显示,猕猴桃体内GalUR蛋白的分子量约37kD。在猕猴桃不同组织和发育过程中,GalUR表达水平与AsA含量表现为高度一致,但前体饲喂发现,与L-半乳糖相比,D-半乳糖醛酸并不能有效促进猕猴桃AsA的合成。这些结果表明以单体形式存在的GalUR与猕猴桃AsA合成有着密切关系,但它可能并不是猕猴桃AsA积累的主要调控环节。

英文摘要：

In present study,D-galacturonate reductase（GalUR）cDNA were cloned from young fruit of Actinidia deliciosa＇Qinmei＇,expressed it in E.coli,and prepared the specific antibody,then we analyzed the relationship between its transcript level and AsA level during fruit development and among different tissues of kiwifruit.The results showed that cDNA of GalUR from young fruit of kiwi had an 990 bp open reading frame（ORF） and encoded a protein of 329 amino acid residues with a putative molecular mass of 37 kD.Its accession No.in GenBank is GU339035.Both of its cDNA and amino acid sequence showed high similarity with GalUR genes reported in other plants,e.g.strawberry.After it was heterologously expressed in E.coli,the fusion protein GalUR-His（His is about 21 kD）was about 58 kD,and existed mainly as inclusion body.Western blotting showed that the antibody prepared in rabbit reacted specifically with protein extracted from kiwifruit tissues,and the size of GalUR protein in kiwifruit wasalso about 37 kD.Although GalUR expression levels had a good consistency with AsA content during fruit development and among different tissues of kiwi,the ratio of AsA produced from D-galacturonate was much lower than that from L-galactose by feeding experiment.These results suggest that GalUR in kiwifruit,as a monomer protein,has a good correlation with AsA content,but it is not a main controlling point in regulating AsA accumulation in kiwifruit.