Cloning of fatty acid desaturase-coding sequence (Lufad3) from flax and its functional validation in rice

Flax contains very high levels of alpha-linolenic acid (57%) and a fatty acid desaturase 3-coding sequence (Lufad3) of flax has been amplified from the RNA isolated from developing seeds. The deduced amino acid sequence of LuFAD3 showed the presence of three histidine motifs, six membrane spanning domains and an endoplasmic reticulum (ER) retention signal KSK, indicating its plausible localization into the ER. Flax is not amenable for genetic transformation and not suitable for functional validation of Lufad3 gene. Hence, rice with well-developed genetic transformation has been selected as heterologous host system. Coding sequence of Lufad3 driven by maize Ubi1 promoter has been introduced into indica rice by Agrobacterium tumefaciens-mediated genetic transformation. Southern analysis of putative transformants (T (0)) revealed signals at variable lengths at > 5 kbp indicating random integration of transgene into the genomes of different transformants. The Mendelian segregation observed for selectable marker gene hyg in both T-1 and T-2 generations confirmed stable inheritance and single-site integration of transgenes. As compared to untransformed control (UC), homozygous transgenic rice expressing Lufad3 showed higher levels of essential alpha-linolenic acid in leaves and seeds validating its functionality.