Cloning Of L-Galactose Dehydrogenase CDNA From Apple And The Construction Of Its Sense And Antisense Expression Vectors

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...