【作者】 肖妮娜；

【导师】 马锋旺；

【作者基本信息】 西北农林科技大学 ， 果树学， 2006， 硕士

【摘要】 苹果(Malus pumila Mill.)属蔷薇科蔷薇属植物。抗坏血酸(AsA)俗称维生素C,在植物抵抗氧化胁迫、增强光合保护、调节细胞的分裂、生长过程以及信号传递等方面都具有非常重要的作用。最近几年对AsA在植物中的功能及AsA生物合成途径研究得较深入,在此基础上,以提高植物中AsA含量为目的的相关基因工程研究将为进行提高农产品中维生素C含量、改善农作物的营养品质、增强其抗逆性等方面的研究打下基础。L-半乳糖脱氢酶是AsA合成的L-半乳糖途径中催化L-半乳糖生成L-半乳糖内酯的关键酶。本试验以嘎拉苹果叶片为材料,通过RT-PCR法克隆了调控AsA合成的L-半乳糖脱氢酶基因,进行了序列分析,并且构建了正义和反义表达载体。为进一步研究该酶的分子结构、功能、表达特点以及通过转化研究植物合成和积累AsA的分子机制奠定了基础。本试验获得的主要结果有:1.用RT-PCR法从苹果叶片中克隆出L-半乳糖脱氢酶的cDNA全长,其全长cDNA序列包含一个长度为972bp的开放阅读框(ORF),编码324个氨基酸。该片段与GenBank中其它植物的L-半乳糖脱氢酶基因核苷酸序列同源性分析表明该序列与猕猴桃的同源性为81%,与辣椒的同源性为80%,与菠菜的同源性为78%。2.借助PMD18-T载体将L-半乳糖脱氢酶基因的完整ORF序列正向插入到植物表达载体pSB166的多克隆位点上,构建了含有GalDH全长cDNA的植物正义表达载体。并经酶切、PCR鉴定,已导入农杆菌。3.将L-半乳糖脱氢酶基因的完整序列反向插入到植物表达载体pSB166的多克隆位点上,构建了反义表达载体,并通过直接转化法将其转化入农杆菌EHA105。更多还原

【Abstract】 Gala (Malus pumila Mill.) is the member of the Rosaceae .L-ascorbic acid (AsA) gose by the name of Vitamin C. It has a lot of function in In recent years, great advances have been made in the functions and the biosynthetic pathways of L-ascorbic acid. Based on that, the ASA related gene engineering research will pave new ways for improving the AsA content of agricultural products, ameliorating crop’s nutrition and enhancing the resistance.L-Galactose dehydrogenase (L-GalDH), the key enzyme in the L-galactose pathw of aascorbate biosynthesis, oxidizes l-Gal to l-galactono-1,4-lactone ( l-GalL).Full-length cDNA encoding L-GalDH was cloned from Royal Gala apple (Malus Pumila Mill cv. Royal Gala) leaves by RT-PCR method.Then target sequence was analized and the sence and antisense plant expression vectors were constructed. The result provided the basis for studying L-GalDH molecular structure, enzyme function, gene expression characteristics and the mechanism of AsA biosynthesis and accumulation by transformation.The main results as follows:1. Full-length cDNA was isolated from Malus pumila Mill cv Royal Gala leaves using RT-PCR method. Then the target fragment was purified from agarose gel and integrated into pMD18-T cloning vector. After being transformed into E.coli.DH5α, the colony assay was carried out by endonuclease restriction and PCR amplification, then the positive clone was sequenced.Sequencing result shows that the full-length of cDNA of apple GalDH has been successfully cloned.Sequence analysis indicated the fragment represents an open reading frame of 976bp encoding 324 amino acids. The nucleic acid sequence analysis showed high homology with the L-GalDH sequence known from other plants. .Comparisons between the- L-GalDH sequences from Actinidia deliciosa,Capsicum annuum,Spinacia oleracea the homology was 81%,80%and78%respectively..2. The plant sence expression vector was constructed by introducing full-length L-GalDH cDNA from pMD18-T cloning vector into binary vector pSB166. The recombinant更多还原