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.
Highlights
Plukenetia volubilis L. is a perennial woody vine of the Euphorbiaceae family, which can produce fruits in the same year of planting and within 2–3 years after it enters the productive period of up to 10 years, with great economic value [1]
Comparison of the PvFAD3 cDNA sequence with the transcript sequence of Unigene0043398 indicated that the PvFAD3 gene contained eight exons, and the genomic DNA fragment was interrupted by seven introns at the ‘GT’ and ‘AG’ sites
Linoleic acid content was significantly lower in positive transgenic tobacco seeds than in wild-type (p < 0.001). These results show that the overexpression of the PvFAD3 in tobacco could significantly enhance the conversion of linoleic acid to α-linolenic acid and increase the oil content of the transgenic tobacco seeds
Summary
Plukenetia volubilis L. is a perennial woody vine of the Euphorbiaceae family, which can produce fruits in the same year of planting and within 2–3 years after it enters the productive period of up to 10 years, with great economic value [1]. With an oil content of 35% to 60% [2], P. volubilis seeds are free from any toxins and harmful substances and contain more than 90% unsaturated fatty acids. Α-Linolenic acid (C18:3, ALA) is an essential, plant-based, ω-3 polyunsaturated fatty acid that is obtained through the diet It is a precursor for synthesizing two essential factors (EPA, C20:5, and DHA, C22:6) in the human body [7,8]. Several studies have reported that FAD3 overexpression can significantly increase α-linolenic acid content in leaves, roots, fruits, seeds, and other tissues of Arabidopsis thaliana [10], Brassica napus [11], and Lycopersicon esculentum [12]. We presumed that the extremely high expression level of PvFAD3 was closely related to the efficient accumulation of α-linolenic acid, which is consistent with the findings of Wang et al [22,23] on P. volubilis seeds
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