Engineering the Staple Oil Crop Brassica napus Enriched with α-Linolenic Acid Using the Perilla FAD2-FAD3 Fusion Gene.

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 FAD2 and FAD3 coding regions from the ALA-king species Perilla frutescens were fused using a newly designed double linker LP4-2A, driven by a seed-specific promoter PNAP, and engineered into a rapeseed elite cultivar ZS10 with canola quality background. The mean ALA content in the seed oil of PNAP:PfFAD2-PfFAD3 (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 PfFAD2-PfFAD3 transgenic rapeseed lines driven by the constitutive promoter PD35S 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 BnFAD2 and BnFAD3.

Molecular Cloning and Characterization of FAD6 Gene from Chia (Salvia hispanica L.).

Plastidial Δ12 fatty acid desaturase (FAD6) is a key enzyme for linoleic acid (LA) and α-linolenic acid (ALA) biosynthesis. Chia (Salvia hispanica L.) is a revived omega-3 plant source that is richest in ALA level. In this study, based on the RACE method, one full-length cDNA sequence encoding FAD6, named ShFAD6, was isolated from chia. There exist three alternative transcription start sites and five alternative poly(A) tailing sites in ShFAD6. The 5'UTR of ShFAD6 contains a purine-stretch of 44 bp. ShFAD6 has an ORF of 1335 bp encoding a 444 aa protein of 51.33 kDa. ShFAD6 contains a conserved Delta12-FADS-like domain together with three strong trans-membrane helices and three histidine motifs. There also exists a chloroplast transmit peptide in ShFAD6 N-terminal. Phylogenetic analyses validated its identity of dicot FAD6 protein and suggested some critical evolutionary features of plant FAD6 genes. Heterologous yeast expression confirmed the catalytic activity of ShFAD6. The qRT-PCR assay showed that ShFAD6 is mainly expressed in leaves, stems, flowers, buds and early-stage seeds, and also responded to various stresses and hormone treatments. Under Sclerotinia infection, qRT-PCR and fluorescence imaging illustrated the possible correlation of ShFAD6 expression and photosynthesis. This study provides insight for further function study of ShFAD6 in oil quality improvement in staple oilseed crops as well as stress response and adaptation in plants.