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

Document Type : Original Article

Authors

Campus of Agriculture and Natural Resources, Razi University, Kermanshah, Iran

10.29252/jabr.05.02.04

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-L-galactose 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.

Keywords

References

  1. Cruz-Rus E, Amaya I, Valpuesta V. The challenge of increasing vitamin C content in plant foods. Biotechnol J. 2012;7(9):1110- 1121. doi:10.1002/biot.201200041.
  2. Gallie DR. L-ascorbic Acid: a multifunctional molecule supporting plant growth and development. Scientifica (Cairo). 2013;2013:795964. doi:10.1155/2013/795964.
  3. Gallie DR. Increasing vitamin C content in plant foods to improve their nutritional value-successes and challenges. Nutrients. 2013;5(9):3424-3446. doi:10.3390/nu5093424.
  4. Hancock RD, Viola R. Biosynthesis and catabolism of L-ascorbic acid in plants. Critical Reviews in Plant Sciences. 2005;24(3):167- 188. doi:10.1080/07352680591002165.
  5. Wheeler GL, Jones MA, Smirnoff N. The biosynthetic pathway of vitamin C in higher plants. Nature. 1998;393(6683):365-369. doi:10.1038/30728.
  6. Li M, Ma F, Liang D, Li J, Wang Y. Ascorbate biosynthesis during early fruit development is the main reason for its accumulation in kiwi. PLoS One. 2010;5(12):e14281. doi:10.1371/journal. pone.0014281.
  7. Bulley SM, Rassam M, Hoser D, et al. Gene expression studies in kiwifruit and gene over-expression in Arabidopsis indicates that GDP-L-galactose guanyltransferase is a major control point of vitamin C biosynthesis. J Exp Bot. 2009;60(3):765-778. doi:10.1093/ jxb/ern327.
  8. Venkatesh J, Park SW. Role of L-ascorbate in alleviating abiotic stresses in crop plants. Bot Stud. 2014;55(1):38. doi:10.1186/1999- 3110-55-38.
  9. Sambrook JR, Russell DW. Molecular cloning: a laboratory manual. Q Rev Biol. 2001;76:348-349.
  10. Linster CL, Gomez TA, Christensen KC, et al. Arabidopsis VTC2 encodes a GDP-L-galactose phosphorylase, the last unknown enzyme in the Smirnoff-Wheeler pathway to ascorbic acid in plants. J Biol Chem. 2007;282(26):18879-18885. doi:10.1074/jbc. M702094200.
  11. Kondo T, Fujikawa Y, Ueda A, et al. Cloning and gene expression analysis of ascorbic acid biosynthesis enzymes in Moringa oleifera. Afr J Agric Sci. 2015;10(22):2274-2285. doi:10.5897/ AJAR2014.9306.
  12. Urzica EI, Adler LN, Page MD, et al. Impact of oxidative stress on ascorbate biosynthesis in Chlamydomonas via regulation of the VTC2 gene encoding a GDP-L-galactose phosphorylase. J Biol Chem. 2012;287(17):14234-14245. doi:10.1074/jbc.M112.341982.
  13. Hemavathi, Upadhyaya CP, Akula N, et al. Enhanced ascorbic acid accumulation in transgenic potato confers tolerance to various abiotic stresses. Biotechnol Lett. 2010;32(2):321-30. doi:10.1007/ s10529-009-0140-0.
Journal of Applied Biotechnology Reports
Volume 5, Issue 2
June 2018
Pages 59-63

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History

  • Receive Date: 25 January 2018
  • Revise Date: 10 March 2018
  • Accept Date: 05 April 2018
  • First Publish Date: 01 June 2018

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