Silencing of GmFAD3 gene by siRNA leads to low α-linolenic acids (18:3) of fad3-mutant phenotype in soybean [Glycine max (Merr.)]

Abstract

RNA interference (RNAi) has been recently employed as an effective experimental tool for both basic and applied biological studies in various organisms including plants. RNAi deploys small RNAs, mainly small interfering RNAs (siRNAs), to mediate the degradation of mRNA for regulating gene expression in plants. Here we report an efficient siRNA-mediated gene silencing of the omega-3 fatty acid desaturase (FAD3) gene family in a complex genome, the soybean (Glycine max). The FAD3 enzyme is responsible for the synthesis of alpha-linolenic acids (18:3) in the polyunsaturated fatty acid pathway. It is this fatty acid that contributes mostly to the instability of soybean and other seed oils. Therefore, a significant reduction of this fatty acid will increase the stability of the seed oil, enhancing the seed agronomical value. A conserved nucleotide sequence, 318-nt in length, common to the three gene family members was used as an inverted repeat for RNA interference. The RNAi expression cassette was driven by a seed-specific promoter. We show that the transgene-produced siRNA caused silencing of FAD3 that was comparable to the fad3 mutant phenotype and, furthermore, that such a silencing is stably inherited in engineered soybean lines. Since the pool size of the alpha-linolenic acids is small relative to the other polyunsaturated fatty acids in soybean, the significant reduction of this fatty acid suggests a role and great potential for the siRNA strategy in silencing gene families in a complex genome.

A Family of Negative Regulators Targets the Committed Step of de Novo Fatty Acid Biosynthesis

“…Inverted repeats targeting At-BADC1 were inserted into the pMU103 vector (Flores et al, 2008). The repeats coded for bases 774 to 1034 of the cDNA sequence (accession AT3G56130.1).…”

Soybean Oil-Quality Variants Identified by Large-Scale Mutagenesis

“…Mutants with low linolenate (designated fan) have been identified previously, and two independent low linolenic mutants have been shown to contain lesions in the FAD3A gene [5,10,31,32]. When all three FAD3 homologs are downregulated with RNAi in seeds, International Journal of Agronomy linolenic acid levels can be further reduced, therefore, it is possible that mutation in FAD3B or FAD3C may also result in low levels of linolenic acid [33]. Eight lines containing 10.5-12.9% linolenic acid were isolated in this study (lines 45-52).…”

Genome Editing with Engineered Nucleases in Economically Important Animals and Plants: State of the Art in the Research Pipeline

“…Examples of the transgenic approach include Golden rice with provitamin A content (Beyer et al, 2002) or soybean with decreased α-linolenic fatty acid content (Flores et al, 2008). Seed oil composition in soybean was also the subject in the study by Haun et al (2014) who have used TALENs to knock-out the fatty acid desaturase 2 (fad2) genes.…”