Abstract
BackgroundThe oilseed Camelina sativa is grown for a range of applications, including for biofuel, biolubricants, and as a source of omega-3 fatty acids for the aquaculture feed industry. The seed meal co-product is used as a source of protein for animal feed; however, the low value of the meal hinders profitability and more widespread application of camelina. The nutritional quality of the seed meal is largely determined by the abundance of specific seed storage proteins and their amino acid composition. Manipulation of seed storage proteins has been shown to be an effective means for either adjustment of nutritional content of seeds or for enhancing accumulation of high-value recombinant proteins in seeds.ResultsCRISPR/Cas9 gene editing technology was used to generate deletions in the first exon of the three homoeologous genes encoding the seed storage protein CRUCIFERIN C (CsCRUC), creating an identical premature stop-codon in each and resulting in a CsCRUC knockout line. The mutant alleles were detected by applying a droplet digital PCR drop-off assay. The quantitative nature of this technique is particularly valuable when applied to polyploid species because it can accurately determine the number of mutated alleles in a gene family. Loss of CRUC protein did not alter total seed protein content; however, the abundance of other cruciferin isoforms and other seed storage proteins was altered. Consequently, seed amino acid content was significantly changed with an increase in the proportion of alanine, cysteine and proline, and decrease of isoleucine, tyrosine and valine. CsCRUC knockout seeds did not have changed total oil content, but the fatty acid profile was significantly altered with increased relative abundance of all saturated fatty acids.ConclusionsThis study demonstrates the plasticity of the camelina seed proteome and establishes a CRUC-devoid line, providing a framework for modifying camelina seed protein composition. The results also illustrate a possible link between the composition of the seed proteome and fatty acid profile.
Highlights
The oilseed Camelina sativa is grown for a range of applications, including for biofuel, biolubricants, and as a source of omega-3 fatty acids for the aquaculture feed industry
Design of the CsCRUC guide RNA (gRNA) spacer sequence and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 construct The C. sativa genome sequence encodes three homoeologues of CRUCIFERIN C (CRUC), which correspond to its three sub-genomes (CsCRUC-G1, CsCRUC-G2, and CsC RUC-G3; for gene identifiers see Fig. 1a) [26]
This study focused on the CRUC homoeologues as this group has the most abundant transcript of the gene family [38] (Additional file 1: Figure S1), as observed in Arabidopsis [36], and is the most divergent at the amino acid level [37, 39], thereby making elimination of CRUC a good target for altering camelina seed protein and amino acid composition
Summary
The oilseed Camelina sativa is grown for a range of applications, including for biofuel, biolubricants, and as a source of omega-3 fatty acids for the aquaculture feed industry. The relative abundance and amino acid content of different seed storage proteins influence the nutritional quality and economic value of the seed meal [13]. Manipulation of seed storage proteins is an area of interest in a number of plant species for improvement of nutrient composition and to express foreign proteins [15, 18,19,20,21,22]. Such efforts are largely constrained by the inherent metabolic programming directing production of endogenous seed storage proteins [17, 19]. Reduction of seed storage proteins using gene knock-out or knock-down approaches has been effective in by-passing these limits and increasing foreign protein yield in soybean [19], Arabidopsis [23] and rice [21, 24] by making available metabolic resources originally monopolized by endogenous seed storage protein synthesis
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