Abstract
Seed oils are used as edible oils and increasingly also for industrial applications. Although high-oleic seed oil is preferred for industrial use, most seed oil is high in polyunsaturated fatty acids (PUFAs) and low in monounsaturated fatty acids (MUFAs) such as oleic acid. Oil from Camelina, an emerging oilseed crop with a high seed oil content and resistance to environmental stress, contains 60% PUFAs and 30% MUFAs. Hexaploid Camelina carries three homoeologs of FAD2, encoding fatty acid desaturase 2 (FAD2), which is responsible for the synthesis of linoleic acid from oleic acid. In this study, to increase the MUFA contents of Camelina seed oil, we generated CsFAD2 knockout plants via CRISPR-Cas9-mediated gene editing using the pRedU6fad2EcCas9 vector containing DsRed as a selection marker, the U6 promoter to drive a single guide RNA (sgRNA) covering the common region of the three CsFAD2 homoeologs, and an egg-cell-specific promoter to drive Cas9 expression. We analyzed CsFAD2 homoeolog-specific sequences by PCR using genomic DNA from transformed Camelina leaves. Knockout of all three pairs of FAD2 homoeologs led to a stunted bushy phenotype, but greatly enhanced MUFA levels (by 80%) in seeds. However, transformants with two pairs of CsFAD2 homoeologs knocked out but the other pair wild-type heterozygous showed normal growth and a seed MUFAs production increased up to 60%. These results provide a basis for the metabolic engineering of genes that affect growth in polyploid crops through genome editing.
Highlights
Plants contain a large proportion of polyunsaturated fatty acids (PUFAs), such as linoleic acid and α-linolenic acid, in their membrane lipids and storage lipids
Camelina plants with one intact CsFAD2 homoeolog grew normally and yielded seed oil with a 60% monounsaturated fatty acids (MUFAs) content. These results suggest that Camelina requires one functional CsFAD2 homoeolog for normal growth and that the number of functional CsFAD2 homoeologs affects MUFA content in seed oil in a dose-dependent manner
By generating CsFAD2 KO using clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9, we increased the MUFA content of Camelina seeds, but this was accompanied by growth inhibition and deterioration of other agronomic traits
Summary
Plants contain a large proportion of polyunsaturated fatty acids (PUFAs), such as linoleic acid and α-linolenic acid, in their membrane lipids and storage lipids. Seed oils are primarily used as edible oils, but their industrial use has been gradually increasing (Biermann et al, 2011). Seed oils have excellent potential for industrial applications because their major constituents are C16-C18 fatty acids, which are similar in chemical structure to diesel oil (Durrett et al, 2008). Monounsaturated fatty acids (MUFAs) such as oleic acid are preferred for industrial uses, for example as biodiesel. High oleic seed oils are favorable for deep frying (Warner and Knowlton, 1997). High-oleic vegetable oil is preferred for both industrial and food uses. Most vegetable oils contain high PUFA and low MUFA levels
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