Molecular cloning and functional characterization of genes involved in the biosynthesis of polyunsaturated fatty acids in oat (Avena sativa L.)

Abstract

This thesis research started with analysis of oat fatty acids by using three different transmethylation methods. Basic sodium methoxide was compared with traditional acidic methanol for the total fatty acid analysis, while diazomethane was used to analyze free fatty acids. Epoxy FAs were readily hydrolyzed to dihydroxy fatty acids under the acidic condition, which suggest an overestimation of hydroxyl fatty acids and underestimation of epoxy fatty acids in previous analyses. The sodium methoxide method proved more reliable to quantify the oat seed fatty acid composition. CDC Dancer oat seed analyzed here was comprised mostly of palmitic acid (PA), oleic acid (OA) and the polyunsaturated fatty acid (PUFA) linoleic acid (LA) in quantities of 23%, 32%, and 37% of total seed FA, respectively. As well, the seed contained small quantities of another PUFA, α-linolenic (ALA) and several unusual oxygenated fatty acids (UFAs), Δ15-hydroxy fatty acid (15HFA) and epoxy fatty acids in quantities of 0.85%, 0.68%, and 2.3%, respectively. This thesis further aimed to identify and assemble all FAD2-like genes from an oat Expressed-Sequence Tag (EST) database using FAD2 and FAD2-like proteins from other organisms as query sequences in order to clone all putative FAD2-like genes-of-interest (GOIs) from oat. From the contig assemblies of retrieved oat ESTs, four distinct, putative genes were identified. From the Δ12-desaturase (FAD2) queries, a putative FAD2-like (AsFAD2) gene was identified; the Δ15-desaturase (FAD3) queries revealed two putative oat FAD3-like (AsFAD3-1 and AsFAD3-2) genes, while an ω-3 desaturase (FAD7) query identified a fourth putative full-length FAD6-like coding sequence of two possible lengths, AsFADX and AsFADX+. The GOIs were then subcloned into a yeast expression vector and functionally characterized. AsFAD2a and AsFAD2b both demonstrated Δ12 desaturation on 18:1-9c substrate. AsFAD3-1 had no activity on any substrates present, while AsFAD3-2 exhibited weak Δ15-desaturation activity specifically on 18:2-9c,12c. Finally, AsFADX converted 18:1-9c to 18:2-9c,12c, while AsFADX+ had no activity. Then, a comparative analysis of transcript levels of these GOIs via quantitative real-time PCR (qRT-PCR) was performed across oat germinating seed, root, leaf, and developing seed. AsFAD2 transcript abundance was generally much higher than AsFAD3-1 and AsFAD3-2 in all tissues. AsFAD3-1 mRNA level was highest in developing seed tissue, slightly lower in leaf tissue, and lowest in root. AsFAD3-2 mRNA was highest in germinating seed, and lowest in leaf tissue. In summary, the data produced from this thesis could be used to enhance breeding efforts for establishing oat cultivars with healthier oil content.