Abstract
BackgroundLinoleic acid is an important polyunsaturated fatty acid, required for all eukaryotes. Microsomal delta-12 (Δ12) oleate desaturase (FAD2) is a key enzyme for linoleic acid biosynthesis. Desert shrub Artemisia sphaerocephala is rich in linoleic acid, it has a large FAD2 gene family with twenty-six members. The aim of this work is to unveil the difference and potentially functionality of AsFAD2 family members.ResultsFull-length cDNAs of twenty-one AsFAD2 genes were obtained from A. sphaerocephala. The putative polypeptides encoded by AsFAD2 family genes showed a high level of sequence similarity and were relatively conserved during evolution. The motif composition was also relatively conservative. Quantitative real-time PCR analysis revealed that the AsFAD2–1 gene was strongly expressed in developing seeds, which may be closely associated with the high accumulating ability of linoleic acid in A. sphaerocephala seeds. Although different AsFAD2 family members showed diverse response to salt stress, the overall mRNA levels of the AsFAD2 family genes was stable. Transient expression of AsFAD2 genes in the Nicotiana benthamiana leaves revealed that the encoded proteins were all located in the endoplasmic reticulum. Heterologous expression in Saccharomyces cerevisiae suggested that only three AsFAD2 enzymes, AsFAD2–1, − 10, and − 23, were Δ12 oleate desaturases, which could convert oleic acid to linoleic acid, whereas AsFAD2–1 and AsFAD2–10 could also produce palmitolinoleic acid.ConclusionsThis research reported the cloning, expression studies, subcellular localization and functional identification of the large AsFAD2 gene family. These results should be helpful in understanding fatty acid biosynthesis in A. sphaerocephala, and has the potential to be applied in the study of plant fatty acids traits.
Highlights
Linoleic acid is an important polyunsaturated fatty acid, required for all eukaryotes
We cloned the full-length Complementary DNA (cDNA) of twenty-one A. sphaerocephala FAD2 (AsFAD2) genes from different A. sphaerocephala tissues using reverse transcription Polymerase chain reaction (PCR) (RT-PCR) and rapid amplification of cDNA ends (RACE) methods based on transcriptome sequence data (Additional file 1: Table S1)
The results showed that seven AsFAD2 cDNA-encoded proteins were localized to network-like organelles, the strong green fluorescent protein (GFP) and red fluorescent protein (RFP) signals were observed in the epidermal cells of tobacco leaves, and the both fluorescent signals could be overlapped and displayed as yellow fluorescent signals, indicating that the selected seven AsFAD2s were transiently expressed in the endoplasmic reticulum (ER) of tobacco leaf epidermal cells (Fig. 5)
Summary
Linoleic acid is an important polyunsaturated fatty acid, required for all eukaryotes. Microsomal delta (Δ12) oleate desaturase (FAD2) is a key enzyme for linoleic acid biosynthesis. Desert shrub Artemisia sphaerocephala is rich in linoleic acid, it has a large FAD2 gene family with twenty-six members. Linoleic acid (LA, C18:2) is known as an important polyunsaturated fatty acid (PUFA), required for normal growth of all eukaryotes [1]. LA has the effect of lowering serum cholesterol and triglyceride levels, which is good for preventing cardiovascular diseases, LA synthesis in plants is usually catalyzed by membrane-bound enzymes Δ12 fatty acid desaturases (FADs), it is known as ω-6 FADs, which act by introducing a double bond at the delta-12 (Δ12) position. Different numbers of the FAD2 genes in plants may be a result of their adaptation to diverse environmental conditions
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