Evening primrose (Oenothera biennis) Δ6 fatty acid desaturase gene family: cloning, characterization, and engineered GLA and SDA production in a staple oil crop

Abstract

γ-Linolenic acid (GLA), stearidonic acid (SDA), and other polyunsaturated fatty acids (PUFAs) play important roles in human health, and Δ6 fatty acid desaturase (D6D) is a key enzyme for PUFA biosynthesis. Oenothera biennis is a traditional GLA-producing oil crop. Here we report the cloning, characterization, and metabolic engineering of its D6D gene family. The intronless 1922-bp ObD6D1 and 1752-bp ObD6D2 both have alternative transcription start sites, alternative polyadenylation sites, and repeated stop codons. Phylogenetic study revealed five large groups of front-end desaturases which are not simply correlated with desaturase types or organism origins. Any type of front-end enzyme has independently originated for multiple times. “Higher plants,” “higher animals,” and cyanobacteria each has front-end enzymes of single ancient origin, but other taxa are contrary. ObD6D1 and ObD6D2 are most abundantly expressed in bud and root, respectively. In yeast expression, ObD6D1 and ObD6D2 both catalyzed linoleic acid into GLA, and α-linolenic acid into SDA. Based on a newly constructed double-selection vector system, four constructs of ObD6D1 and ObD6D2 both individually driven by double CaMV35S promoter PD35S and seed-specific promoter PNAP were transformed into Brassica napus via Agrobacterium. T1 plants, T2 elite lines, and homozygous T3 elite lines accumulated average GLA + SDA levels of 6.04, 15.77, and 21.56% of seed fatty acids, respectively. ObD6D2 showed higher effect than ObD6D1. PD35S-D6D engineering has little side effect but is weaker than PNAP-D6D engineering in terms of GLA + SDA productivity.