Extending Omega 3/6 Fatty Acid Pathway in Arabidopsis thaliana using Microalgal Gene Δ6Des for Stearidonic acid and Dihomo-γ-linolenic acid Production

Abstract

• Microalgal gene Δ6Des was cloned in a binary vector pCAMBIA1303. • F 1 transgenic plants having Δ6Des were viable at a concentration of 25 mg/L of hygromycin medium • Transgenic Arabidopsis thaliana showed novel ω3/6 fatty acids SDA (0.58%) and DGLA (25. 34%) To enhance the quality and quantity of oils in plant systems, specific candidate genes such as desaturase and elongase that can extend the biosynthetic pathways of very long chain poly unsaturated fatty acids (VLCPUFAs) are essential. In general, plant systems are capable of synthesizing essential fatty acids (EFAs). However, incorporation of a preliminary gene, Δ6Desaturase ( Δ6Des ), in the fatty acid biosynthetic pathway will elongate the pathway further to produce successive fatty acids. In this study, Δ6Des gene extracted from the marine microalga Isochrysis sp. was used to extend the omega (ω) 3/6 fatty acid biosynthetic pathway in a model plant Arabidopsis thaliana using Agrobacterium -mediated genetic transformation. Hygromycin-resistant plants were (25 mg/L) used for GUS and GFP assays and for molecular confirmation. GUS assay showed an intense blue color in F 1 transgenic A. thaliana plant tissues grown in the selection medium, which confirmed the presence of GUS gene in the T-DNA region, whereas non-transformed plant tissues such as leaf or stem did not show any blue color. GFP analysis also showed a green fluorescent color in tissues such as leaf, stem, and floral parts of F1 transgenic A. thaliana plants, which confirmed the presence of T-DNA. PCR and Southern blotting analyses also clearly showed the presence of Δ6Des in the T-DNA region of the F 1 transgenic plants. The fatty acid profile of F 1 transgenic plants showed the production of new fatty acids such as stearidonic acid (SDA-0.58%) and dihomo-γ-linolenic acid (DGLA-25.34%) that were not observed in non-transformed control plants. All these results showed the synthesis of new fatty acids in the transgenic plant, which proved that PUFA biosynthetic pathway can be extended in oilseed plants if specific genes are introduced through genetic transformation. This is the first study to report the productions of new fatty acids like SDA and DGLA in A. thaliana using Δ6Des gene from marine microalga Isochrysis sp.