Isolation and Characterization of GDP-L-Galactose Phosphorylase Gene of Vitamin C Biosynthesis Pathway from Kiwi

Abstract

Introduction: Vitamin C is a major antioxidant in plants and plays an important role in reducing the activity of reactive oxygen species. In humans, the main role of this molecule is the elimination of activity of active oxygen species along with being cofactor for many enzymes. Human is one of the few mammalian species that can not synthesize this vitamin and needs to get it through food sources. The GDP-Lgalactose phosphorylase (GGP) gene is one of the most important genes in the biosynthetic pathway of vitamin C, which codes for the GDPL-galactose phosphorylase enzyme. Isolation of GGP gene is an important step in transferring it to elevate vitamin C biosynthesis in plants. Materials and Methods: In current study, the isolation of this gene from kiwi plant was carried out and then was cloned in the pTG19-T plasmid via T/A cloning and subsequently sequenced to confirm it. Results: Sequencing analysis of the GGP gene showed that this fragment contains 1383 bp and the start and stop codons were ATG and TGA, respectively. The bioinformatics analysis of this gene can provide important information on gene and protein structure. The alignment of cloned sequence was done with other Actinidia DNA sequences. The results based on neighbor-joining alignment showed that some of the mutations in nucleotides were related to the third nucleotide in a specific codon. Also, the minimum distance of protein sequences was observed between isolated GGP and Actinidia chinensis. Conclusions: Based on analyses, isolated gene (GGP) can be used for increase vitamin C content in other plants such as cucumber and for resistance to environmental stresses in different plants.