Abstract
Omega-3 fatty acid desaturase (ω-3 FAD, D15D) is a key enzyme for α-linolenic acid (ALA) biosynthesis. Both chia (Salvia hispanica) and perilla (Perilla frutescens) contain high levels of ALA in seeds. In this study, the ω-3 FAD gene family was systematically and comparatively cloned from chia and perilla. Perilla FAD3, FAD7, FAD8 and chia FAD7 are encoded by single-copy (but heterozygous) genes, while chia FAD3 is encoded by 2 distinct genes. Only 1 chia FAD8 sequence was isolated. In these genes, there are 1 to 6 transcription start sites, 1 to 8 poly(A) tailing sites, and 7 introns. The 5’UTRs of PfFAD8a/b contain 1 to 2 purine-stretches and 2 pyrimidine-stretches. An alternative splice variant of ShFAD7a/b comprises a 5’UTR intron. Their encoded proteins harbor an FA_desaturase conserved domain together with 4 trans-membrane helices and 3 histidine boxes. Phylogenetic analysis validated their identity of dicot microsomal or plastidial ω-3 FAD proteins, and revealed some important evolutionary features of plant ω-3 FAD genes such as convergent evolution across different phylums, single-copy status in algae, and duplication events in certain taxa. The qRT-PCR assay showed that the ω-3 FAD genes of two species were expressed at different levels in various organs, and they also responded to multiple stress treatments. The functionality of the ShFAD3 and PfFAD3 enzymes was confirmed by yeast expression. The systemic molecular and functional features of the ω-3 FAD gene family from chia and perilla revealed in this study will facilitate their use in future studies on genetic improvement of ALA traits in oilseed crops.
Highlights
In the family Lamiaceae, chia (Salvia hispanica, 2n = 12) and perilla (Perilla frutescens, 2n = 40), which are annual herbaceous plants, are 2 rich sources of ω-3 polyunsaturated fatty acids (PUFAs)
We isolated 2 heterozygous alleles containing some SNPs for PfFAD3, PfFAD7 and PfFAD8 from perilla, whereas 2 distinct ShFAD3 genes, 2 heterozygous ShFAD7 alleles, and 1 ShFAD8 gene were cloned from chia (S1 Fig; Table 1)
Similar to the situation of mRNA analysis, we found that ShFAD7a/b had higher identities with PfFAD7a/b than with ShFAD8, whereas ShFAD8 showed higher similarity to PfFAD8a/b than to ShFAD7a/b
Summary
In the family Lamiaceae, chia (Salvia hispanica, 2n = 12) and perilla (Perilla frutescens, 2n = 40), which are annual herbaceous plants, are 2 rich sources of ω-3 polyunsaturated fatty acids (PUFAs). Chia and perilla seeds contain 25~40% oil, and ɑ-linolenic acid (ALA, 18:3Δ9,12,15) proportion in their seed oil is the highest among crop sources (about 60~71%) [5,6,7]. ALA is known as an essential fatty acid (FA) for the human daily diet because ALA cannot be synthesized in the human body due to the absence of the ω-3 FAD gene [8]. With respect to the ALA biosynthesis pathway, a small portion of pivotal genes has been described in perilla [12,13,14,15], but genetic and molecular studies are relatively rare for chia [16]. Systemic cloning of the ω-3 FAD gene family of both chia and perilla was performed in this study, which is the first report to provide the full-length cDNA and genomic DNA (gDNA) sequences of chia ω-3 FAD genes, as well as the full-length gDNA sequences of perilla ω-3 FAD genes
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