Transcriptomic and lipidomic analysis reveal molecular mechanisms in the biosynthesis of nervonic acid in engineered Brassica napus seeds

Nervonic acid (NA) is a very long-chain monounsaturated fatty acid with significant medicinal value, but its further development and application are limited by resource scarcity. In our previous studies, metabolic engineering of Brassica napus (B. napus) using multi-gene vectors (Napin-3 and Napin-5) successfully achieved high levels of NA production, with transgenic lines accumulating NA up to 45% of total fatty acids. This study utilizes integrated transcriptomics and lipidomics at 20 and 40 days after flowering (DAF) to elucidate the molecular basis of NA accumulation in transgenic B. napus seeds expressing the CgKCS gene. Integrated transcriptomic and lipidomic analyses revealed that NA biosynthesis predominantly occurs during late seed development and is sequestered into triacylglycerol (TGs), contributing to the increased TG (24:1_18:2_18:3). Transcriptomic analysis identified numerous differentially expressed genes (DEGs) associated with lipid metabolism and seed development. Notably, the expression of several endogenous genes involved in fatty acid elongation and glycerolipid biosynthesis was significantly altered in transgenic seeds. In particular, endogenous KCS6 and KCS19, which are associated with very-long-chain fatty acid elongation, were downregulated, whereas GPAT6 and LPAAT1, key enzymes in glycerolipid assembly, were upregulated. Our findings demonstrate that high-level NA production in B. napus is sustained not only by heterologous gene activity but also by the coordinated response of endogenous acyltransferases and the suppression of competing elongation pathways. These results provide new mechanistic insights into the metabolic plasticity of oilseed crops and identify key genetic targets for further optimizing the production of rare fatty acids.