Overexpression of BnaA08.NDPK Enhances Seed Oil Content and Modulates Fatty Acid Composition in Transgenic Arabidopsis: Insights from Comparative Transcriptomics and Co-Expression Network Analysis

Background/Objectives: Rapeseed (Brassica napus L.) is a major oilseed crop, and enhancing seed oil accumulation is a key breeding objective. However, the transcriptional regulation underlying natural variation in seed oil accumulation and fatty acid composition remains unclear. This study aimed to identify key lipid-associated modules and candidate genes and validate a selected gene. Methods: Twelve rapeseed lines with contrasting oil content were classified into high-, medium-, and low-oil groups. Phenotypic traits were evaluated across field seasons. Fatty acid composition was determined by gas chromatography with flame ionization detection (GC-FID). Transcriptome sequencing was performed on 30-day-after-pollination (DAP) developing seeds, followed by differential expression analysis and weighted gene co-expression network analysis (WGCNA). A candidate gene, BnaA08.NDPK (nucleoside diphosphate kinase), was screened and validated by qRT-PCR. For heterologous functional characterization, BnaA08.NDPK was overexpressed in Arabidopsis, and separate CRISPR/Cas9-edited Arabidopsis lines with disrupted endogenous AtNDPK were generated. Results: Five major fatty acids (C16:0, C18:0, C18:1n9c, C18:2n6c, and C18:3) accounted for >90% of total fatty acids. High-oil materials had higher C18:1n9c but lower C18:2n6c and C18:3, and lower desaturation ratios, than low-oil materials. Up-regulated differentially expressed genes (DEGs) in high-oil lines were enriched in fatty acid biosynthesis, elongation, and glycerolipid metabolism, while down-regulated DEGs were linked to fatty acid degradation and phenylpropanoid biosynthesis. WGCNA identified MEturquoise (positive) and MEgreen (negative) modules correlated with oil content. BnaA08.NDPK was highly expressed in high-oil lines and positively correlated with oil content. In transgenic Arabidopsis, overexpression of BnaA08.NDPK increased seed size, thousand-seed weight, and oil content, with higher C18:1n9c and lower C18:2n6c, whereas CRISPR/Cas9-edited Arabidopsis lines with disrupted AtNDPK showed the opposite trends. Conclusions: Heterologous functional characterization in Arabidopsis supports a positive role of BnaA08.NDPK in seed development and oil accumulation. These findings identify BnaA08.NDPK as a promising candidate gene for improving seed oil accumulation and fatty acid composition, and provide a valuable framework for gene discovery and molecular breeding in rapeseed.