Characteristics of Metabolites by Seed-Specific Inhibition of FAD2 in Brassica napus L

Fatty acid desaturase-2 (FAD2) is a key enzyme in the production of polyunsaturated fatty acids in plants. RNAi technology can reduce the expression of FAD2 genes in Brassica napus seeds and acquire transgenic B. napus plants with a high oleic acid content, but the effect of seed-specific inhibition of FAD2 expression on B. napus seed metabolites is not clear. Here we use widely targeted metabolomics to investigate the metabolites of normal-oleic-acid rapeseed (OA) and high-oleic-acid rapeseed (HOA) seeds, resulting in a total of 726 metabolites being detected. Among them, 24 differential metabolites were significantly downregulated and 88 differential metabolites were significantly upregulated in HOA rapeseed. In further lipid profile experiments, more lipids in B. napus seeds were accurately quantified. The contents of glycolipids and phospholipids that contain C18:1 increased significantly and C18:2 decreased because FAD2 expression was inhibited. The changes in the expression of key genes in related pathways were also consistent with the changes in metabolites. The insertion site of the ihpRNA plant expression vector was reconfirmed through genomewide resequencing, and the transgenic event did not change the sequence of FAD2 genes. There was no significant difference in the germination rate and germination potential between OA and HOA rapeseed seeds because the seedspecific ihpRNA plant expression vector did not affect other stages of plant growth. This work provides a theoretical and practical guidance for subsequent molecular breeding of high OA B. napus.

Automated summary

In this study, widely targeted metabolomics was used to investigate the metabolites of normal-oleic-acid and high-oleic-acid rapeseed, revealing key differential metabolites. Lipid profile experiments further confirmed the impact of RNAi technology on FAD2 gene expression. Additionally, resequencing analysis of the transgenic B. napus plants was conducted, focusing on the insertion site and sequence integrity of FAD2 genes.