Overexpression of PvFAD3 Gene from Plukenetia volubilis Promotes the Biosynthesis of α-Linolenic Acid in Transgenic Tobacco Seeds

Abstract

The ω-3 fatty acid desaturase (FAD3) gene encodes a rate-limiting enzyme in the synthesis of α-linolenic acid. In this study, homologous cloning was used to obtain the full-length sequence of the PvFAD3 gene of Plukenetia volubilis. The full-length DNA sequence was 1871 bp long, with 8 exons and 7 introns. The structural analysis of the amino acid sequence revealed that the PvFAD3 protein contained three histidine-conserved regions and an endoplasmic reticulum retention signal. The real-time reverse transcription-polymerase chain reaction performed for determining the expression patterns of the PvFAD3 gene in different tissues of P. volubilis showed that PvFAD3 expression was highly expressed in the fast oil accumulation stage of seed. The analysis of subcellular localization assay in epidermal cells of tobacco (Nicotiana benthamiana) leaves showed that the PvFAD3 protein was mainly localized in the endoplasmic reticulum. Seed-specific overexpression vectors were constructed, and Agrobacterium-mediated genetic transformation was performed to obtain transgenic tobacco plants overexpressing PvFAD3. The results of fatty acid assays performed using harvested seeds showed a significant increase in α-linolenic acid content, a dramatic decrease in linoleic acid content, and an obvious increase in oil content in transgenic tobacco seeds. Collectively, the PvFAD3 gene of P. volubilis was confirmed as a key enzyme gene for α-linolenic acid synthesis; thus, indicating that the PvFAD3 gene can be used for fatty acid fraction improvement in oilseed plants.