Modern people generally suffer from α-linolenic acid (ALA) deficiency, since most staple food oils are low in ALA content. Thus, the enhancement of ALA in staple oil crops is of importance. In this study, the and coding regions from the ALA-king species were fused using a newly designed double linker LP4-2A, driven by a seed-specific promoter P, and engineered into a rapeseed elite cultivar ZS10 with canola quality background. The mean ALA content in the seed oil of P:- (N23) T5 lines was 3.34-fold that of the control (32.08 vs 9.59%), with the best line being up to 37.47%. There are no significant side effects of the engineered constructs on the background traits including oil content. In fatty acid biosynthesis pathways, the expression levels of structural genes as well as regulatory genes were significantly upregulated in N23 lines. On the other hand, the expression levels of genes encoding the positive regulators of flavonoid-proanthocyanidin biosynthesis but negative regulators of oil accumulation were significantly downregulated. Surprisingly, the ALA level in - transgenic rapeseed lines driven by the constitutive promoter P was not increased or even showed a slight decrease due to the lower level of foreign gene expression and downregulation of the endogenous orthologous genes and .