Increasing vitamin E content of canola (Brassica napus L.) by transferring γ-tmt gene

Document Type: Research paper

Authors

Biotechnology Department, Faculty of Agriculture and Natural Sciences, Imam Khomeini International University, P. O. Box: 34149-16818, Qazvin, Iran.

10.30479/ijgpb.2020.12410.1260

Abstract

Vitamin E is one of the lipid soluble vitamins consisting of several isoforms, including tocopherols and tocotrienols amongst which the alpha tocopherol is the most active one. The conversion of γ tocophrol to α tocopherol takes place by the activity of γ-tmt enzyme. Many plants including canola lack the γ-tmt gene to enable them to convert γ tocophrol into α tocopherol. The aim of this study was to transfer γ-tmt gene into canola plants to enable them to produce α tocopherol and increase their vitamin E content. γ-tmt gene was isolated from tomato (Lycopersicum esculentum L.), Memory1 cultivar. Then, it was amplified using PCR, digested by XbaΙ enzyme, cloned into the pBluescriptΙΙ cloning vector and subcloned into E. coli. The gene was then transferred into pBI121 vector and subsequently the vector containing the γ-tmt gene was transferred into Agrobactearium tumefaciens. Two canola cultivars Zafam and Hayola 401 were used. The cotyledons of canola seeds were inoculated by Agrobactearium tumefaciens. Seven putative transformants from each cultivar (Zarfam 2 to 8 and Hayola 2 to 8) were chosen for further investigations. After the emergence of shoots and roots, the seedlings were assayed for the presence of γ-tmt gene, by PCR. Vitamin E content of the transformed plants was assayed by FTIR spectrophotometer. Results showed several fold increases in vitamin E contents of the transgenic plants compared to the control. The increases in α-tocopherol content were 2.61 and 2.71 times in Zarfam 6 and Hayola 8, respectively. This approach could be considered as a useful method for fortifying oil seed crops with vitamin E.

Keywords


Arun M., Subramanyam K., Theboral J., Sivanadhan G., Rajesh M., Dev G. K., Jagatath B., Manickavasagam M., Girja S., and Ganapathi A. (2014). Transfer and targeted overexpression of γ-tocopherol methyltransferase (γ-tmt) gene using seed-specific promoter improves tocopherol composition in Indian soybean cultivars. Applied Biochemistry and Biotechnology, 172: 1763–1776.

Block M., Brouwer D., and Tenning P. (1989). Transformation of Brassica napus and Brassica oleracea using Agrobacterium tumefaciens and the expression of the bar and neo genes in the transgenic plants. Plant Physiology, 91: 694–701.

Bruni R., Guerrini A., Scalia S., Romagnoli C., and Sacchetti G. (2002). Rapid techniques for the extraction of vitamin E isomers from Amaranthus caudatus seeds: ultrasonic and supercritical fluid extraction. Phytochemical Analysis, 13: 257–261.

Cardoza V., and Stewart C. N. (2003). Increased Agrobacterium-mediated transformation and rooting efficiencies in canola (Brassica napus L.) from hypocotyl segment explants. Plant Cell Reports, 21: 599–604.

Cavell A. C., Lydiate D. J., Parkin I. A. P., Dean C., and Trick M. (1998). Collinearity between a 30-centimorgan segment of Arabidopsis thaliana chromosome 4 and duplicated regions within the Brassica napus genome. Genome, 41: 62–69.

Chen D. F., Zhang M., Wang Y. Q., and Chan X. W. (2012). Expression of γ-tocopherol methyltransferase gene from Brassica napus increased α-tocopherol content in soybean seed. Biologia Plantarum, 56: 131–134.

D’Harlingue A., and Camara B. (1985). Plastid enzymes of terpenoid biosynthesis. Purification and characterization of gamma-tocopherol methyltransferase from Capsicum chromoplasts. Journal of Biological Chemistry, 260: 15200–15203.

Fryer M. J. (1992). The antioxidant effects of thylakoid vitamin E (α-tocopherol). Plant Cell Environment, 15: 381–392.

Groff J. L., Gropper S. S., and Hunt S. M. (1995). The Fat Soluble Vitamins. In: advanced nutrition and human metabolism. Minneapolis: West Publishing Company, 284–324.

Heidari Japelaghi R., Haddad R., and Garoosi G. A. (2011). Rapid and efficient isolation of high quality nucleic acids from plant tissues rich in polyphenols and polysaccharides. Molecular Biotechnology, 49: 129–137.

Herbers K. (2003). Vitamin production in transgenic plants. Journal of Plant Physiology, 160: 821–829. 

Chimire B. K., Seong E. S., Lee C. O., Lim J. D., Lee J. G., Yoo J. H., Chung I. M., Kim N. Y., and Yu C. Y. (2011). Enhancement of α tocopherol content in transgenic Perillafrutescens containing the γTMT gene. African Journal of Biotechnology, 10: 2430–2439.

Koch M., Lemke R., Heise K. P., and Mock H. P. (2003). Characterization of γ–tocopherol methyltransferase from Capsicum annuum L. and Arabidopsis thaliana. European Journal of Biochemistry, 270: 84–92.

Konda A. R., Nazarenus T. J., Nguyen H., Yang J., Gelli M., Swenson S., Shipp J. M., Schmidt M. A., Cahoon R. E., Ciftci O. N., Zhang C., Clemente T. E., and Cahoon E. B. (2020). Metabolic engineering of soybean seeds for enhanced vitamin E tocochromanol content and effects on oil antioxidant properties in polyunsaturated fatty acid-rich germplasm. Metabolic Engineering, 57: 63–73.

Lam Y. T., Stocker R., and Dawes I. W. (2010). Thelipophilic antioxidants alpha-tocopherol and coenzyme Q10 reduce the replicative lifespan of Saccharomyces cerevisiae. Phytochemical Analalysis, 49: 237–44.

Lee B. K., Kim S. L., Kim K. H., Yu S. H., Lee S. C., Zhang Z., Kim M. S., Park H. M., and Lee J. Y. (2008). Seed specific expression of perilla γ-tocopherol methyltransferase gene increases α-tocopherol content in transgenic perilla (Perilla frutescens). Plant Cell, Tissue and Organ Culture, 92: 47–54.

Li D., Sun X., Cui Z., Zhang Y., Bai Y., Wang X., and Chen W. (2015). Differential transformation efficiency of Japonica rice varieties developed in northern China. Crop Breeding and Applied Biotechnology, 15: 162–168.

McKersie B., Hoekstra F. A., and Krieg L. C. (1990). Differences in the susceptibility of plant membrane lipids to peroxidation. Biochimicaet Biophysica Acta, 1030: 119–126.

Me`ne-Saffrane L., and Pellaud S. (2017). Current strategies for vitamin E biofortification of crops. Current Opinion in Biotechnology, 44: 189–197.

Mishra M., Jalil S. Y., Sharma N., and Hudedamani U. (2014). An Agrobacterium mediated transformation system of guava (Psidium guayava L.) with endochitinase gene. Crop Breeding and Applied Biotechnology, 14: 232–237.

Moloney M. M., Walker J. M., and Sharma K. K. (1989). High efficiency tranformation of Brassica napus using Agrobacterium vectors. Plant Cell Reports, 8: 238–42.

Purkrtova Z., Jolivet P., Miquel M., and Chardot T. (2008). Structure and function of seed oleosins and caleosin. Comptes Rendus Biologies, 331: 746–754.

Sambrook J., and Russell D. W. (2001). Molecular Cloning: a laboratory manual. 3nd ed. Vol: 1-3, Cold Spring Harbor Laboratory, Press Cold Spring Harbor New York, USA.

Sattler S. E., Cheng Z., and Della Penna D. (2004). From Arabidopsis to agriculture: Engineering improved vitamin E content in soybean. Trends Plant Science, 9: 365–367.

Shintani D., and Della Penna D. (1998). Elevating the vitamin E content of plants through metabolic engineering. Science, 282(20): 98–100.

Sovero M. (1993). Rapeseed, a new oilseed crop for the United States. In: Janick, J., Simon, J, E., (eds) New crops. Wiley, NewYork, 302–307.

Tavva V. S., Kim Y. H., Kagan I. A., Dinkins R. D., Kim K. H., and Collins G. B. (2007). Increased α-tocopherol content in soybean seed overexpressing the Perilla frutescens γ-tocopherol methyltransferase gene. Plant Cell Reports, 26: 61–70.

Tucker J. M., and Townsend D. M. (2005). Alpha-tocopherol: roles on prevention and therapy of human disease. Biomed Pharmacotherapy, 59: 380– 387.

Zhang G. Y., Liu R. R., Xu G., Zhang P., Li Y., Tang K. X., Liang G. H., and Liu Q. Q. (2013). Increased α tocoterinol content in seeds of transgenic rice overexpressing Arabidopsis γ-tocopherol methyltransferase. Transgenic Research, 22: 89–99.

Zhu Q., Wang B., Tan J., Liu T., Li L., and Liu Y.-G. (2020). Plant synthetic metabolic engineering for enhancing crop nutritional quality. Plant Communications, 1(1): pp. 18. https://doi.org/10.1016/j.xplc.2019.100017.