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Development and characterization of low ?-linolenic acid Brassica oleracea lines bearing a novel mutation in a 'class a' FATTY ACID DESATURASE 3 gene.

ABSTRACT: BACKGROUND: Traditional canola (Brassica napus L.; AACC, 2n=38) cultivars yield seed oil with a relatively high proportion of ?-linolenic acid (ALA; C18:3cis?9,12,15), which is desirable from a health perspective. Unfortunately, due to the instability of this fatty acid, elevated levels also result in oils that exhibit a short shelf life and problems associated with use at high temperatures. As a result, the development of cultivars bearing reduced amounts of ALA in their seeds is becoming a priority. To date, several low ALA B. napus cultivars (~2-3% ALA of total fatty acids) have been developed and molecular analyses have revealed that the low ALA phenotype of lines tested thus far is a result of mutations within two 'class b' FATTY ACID DESATURASE 3 (FAD3) genes. Since B. napus possesses six FAD3 genes (two 'class a', two 'class b' and two 'class c') and ALA levels of approximately 2-3% remain in these low ALA lines, it is likely that the mutation of additional FAD3 genes could further decrease the content of this fatty acid. RESULTS: In this study, we generated low ALA (?2%) lines of B. oleracea, which is the C genome progenitor species of B. napus, via ethyl methanesulphonate (EMS) mutagenesis. We identified a novel nonsense mutation within the 'class a' FAD3 gene (BoFAD3-2) in these lines, which would result in the production of an encoded protein lacking 110 amino acids at its C terminus. When expressed in Saccharomyces cerevisiae, this mutant protein exhibited a drastic decline in its ?-15 desaturase activity compared to the wild-type (wt) protein. Furthermore, we demonstrated that the expression of the mutant BoFAD3-2 gene was significantly reduced in developing seeds of low ALA lines when compared to expression in wt plants. CONCLUSIONS: Given the additive nature of FAD3 mutations on ALA content and the ease with which B. napus can be re-synthesized from its progenitor species, the mutant isolated here has the potential to be used for the future development of B. napus cultivars exhibiting further reductions in ALA content.

SUBMITTER: Singer SD

PROVIDER: S-EPMC4236532 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Development and characterization of low α-linolenic acid Brassica oleracea lines bearing a novel mutation in a 'class a' FATTY ACID DESATURASE 3 gene.

Singer Stacy D SD Weselake Randall J RJ Rahman Habibur H

BMC genetics 20140829

<h4>Background</h4>Traditional canola (Brassica napus L.; AACC, 2n=38) cultivars yield seed oil with a relatively high proportion of α-linolenic acid (ALA; C18:3cis∆9,12,15), which is desirable from a health perspective. Unfortunately, due to the instability of this fatty acid, elevated levels also result in oils that exhibit a short shelf life and problems associated with use at high temperatures. As a result, the development of cultivars bearing reduced amounts of ALA in their seeds is becomin ...[more]

PMID: 25167929

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Project description:High levels of linolenic acid (80 g kg(-1)) are associated with the development of off-flavors and poor stability in soybean oil. The development of low linolenic acid lines such as RG10 (20 g kg(-1) linolenic acid) can reduce these problems. The level of linolenic acid in seed oil is determined by the activities of microsomal omega-3 fatty acid desaturases (FAD3). A major linolenic acid QTL (>70% of variation) on linkage group B2 (chromosome Gm14) was previously detected in a recombinant inbred line population from the RG10 × OX948 cross. The objectives of this study were to validate the major linolenic acid QTL in an independent population and characterize all the soybean FAD3 genes. Four FAD3 genes were sequenced and localized in RG10 and OX948 and compared to the genes in the reference Williams 82 genome. The FAD3A gene sequences mapped to the locus Glyma.14g194300 [on the chromosome Gm14 (B2)], which is syntenic to the FAD3B gene (locus Glyma.02g227200) on the chromosome Gm02 (D1b). The location of the FAD3A gene is the same as was previously determined for the fan allele, that conditions low linolenic acid content and several linolenic acid QTL, including Linolen 3-3, mapped previously with the RG10 × OX948 population and confirmed in the PI 361088B × OX948 population as Linolen-PO (FAD3A). The FAD3B gene-based marker, developed previously, was mapped to the chromosome Gm02 (D1b) in a region containing a newly detected linolenic acid QTL [Linolen-RO(FAD3B)] in the RG10 × OX948 genetic map and corresponds well with the in silico position of the FAD3B gene sequences. FAD3C and FAD3D gene sequences, mapped to syntenic regions on chromosomes Gm18 (locus Glyma.18g062000) and Gm11 (locus Glyma.11g227200), respectively. Association of linolenic acid QTL with the desaturase genes FAD3A and FAD3B, their validation in an independent population, and development of FAD3 gene-specific markers should simplify and accelerate breeding for low linolenic acid soybean cultivars.

Project description:BackgroundDelta-6 desaturase (FADS6) is a key bifunctional enzyme desaturating linoleic acid (LA) or α-linolenic acid (ALA) in the biosynthesis of polyunsaturated fatty acids (PUFAs). In previous work, we analyzed the substrate specificity of two FADS6 enzymes from Mortierella alpina ATCC 32222 (MaFADS6) and Micromonas pusilla CCMP1545 (MpFADS6), which showed preference for LA and ALA, respectively. We also clarified the PUFA profiles in M. alpina, where these lipids were synthesized mainly via the ω6 pathway and rarely via the ω3 pathway and as a result contained low ALA and eicosapentaenoic acid (EPA) levels.ResultTo enhance EPA production in M. alpina by favoring the ω3 pathway, a plasmid harboring the MpFADS6 gene was constructed and overexpressed in a uracil-auxotrophic strain of M. alpina using the Agrobacterium tumefaciens-mediated transformation (ATMT) method. Our results revealed that the EPA production reached 80.0 ± 15.0 and 90.4 ± 9.7 mg/L in MpFADS6 transformants grown at 28 and at 12 °C, respectively. To raise the level of ALA, free form fatty acid was used as exogenous substrate, which increased the EPA production up to 114.5 ± 12.4 mg/L. To reduce the cost of EPA production in M. alpina, peony seed oil (PSO) and peony seed meal (PSM) were used as source of ALA, and EPA production was improved to 149.3 ± 7.8 and 515.29 ± 32.66 mg/L by supplementing with 0.1 % PSO and 50 g/L PSM, respectively. The EPA yield was further increased to 588.5 ± 29.6 mg/L in a 5-L bioreactor, which resulted in a 26.2-fold increase compared to EPA production in wild-type M. alpina. In this work, we have significantly enhanced EPA production through overexpression of a FADS6 desaturase with preference for ALA, combined with supplementation of its substrate.ConclusionAn ALA-preferring FADS6 from M. pusilla CCMP1545 was applied to enhance EPA production in M. alpina. By exogenous addition of peony seed oil or peony seed meal, EPA production was further increased in flasks and fermenters. This research also highlights the value of peony seed meal which can be converted to a high value-added product containing EPA, and as a way to increase the EPA/AA ratio in M. alpina.

Dataset Information

Development and characterization of low ?-linolenic acid Brassica oleracea lines bearing a novel mutation in a 'class a' FATTY ACID DESATURASE 3 gene.

Publications

Development and characterization of low α-linolenic acid Brassica oleracea lines bearing a novel mutation in a 'class a' FATTY ACID DESATURASE 3 gene.

Similar Datasets

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