Many plant species store triacylglycerols (TAGs) in their seeds as a carbon reserve. These TAGs are the major source of energy and carbon material that supports seedling development during the early stages of plant life. Vegetable oils from soybean (Glycine max), Brassica (Brassica napus or B. rapa), sunflower (Helianthus annuus) and many other oilseed crops are also an important source of oil for the human diet or industrial applications including, but not limited to biofuels, biolubricants, nylon precursors, and detergent feedstocks. The degree and/or amount of polyunsaturated fatty acids of vegetable oils are characteristic and determinative properties with respect to oil uses in food or non-food industries. More specifically, the characteristic properties and utilities of vegetable oils are largely determined by their fatty acyl compositions in TAG.
Major vegetable oils are comprised primarily of palmitic (16:0), stearic (18:0), oleic (18:1cis Δ9), linoleic (18:2cis Δ9,12), and α-linolenic (18:3cis Δ9,12,15 or C18:3) acids. Palmitic and stearic acids are, respectively, 16 and 18 carbon-long, saturated fatty acids. Oleic, linoleic, and linolenic acids are 18-carbon-long, unsaturated fatty acids containing one, two, and three double bonds, respectively. Oleic acid is referred to as a monounsaturated fatty acid, while linoleic and linolenic acids are referred to as polyunsaturated fatty acids. Modifications of the fatty acid compositions have been sought after for at least a century in order to provide optimal oil products for human nutrition and chemical (e.g., oleochemical) uses (Gunstone, 1998, Prog Lipid Res 37:277; Broun et al., 1999, Annu Rev Nutr 19:107; Jaworski et al, 2003, Curr Opin Plant Biol 6:178). In particular, the polyunsaturated fatty acids (18:2 and 18:3) have received considerable attention because they are major factors that affect nutritional value and oil stability. However, while these two fatty acids provide essential nutrients for humans and animals, they increase oil instability because they comprise multiple double bonds that may be easily oxidized during processing and storage.
The desaturation of 18:1 into 18:2 is a critical step for synthesizing polyunsaturated fatty acids. During storage lipid biosynthesis, this reaction is known to be catalyzed by the fatty acid desaturase, FAD2, a membrane-bound enzyme located on the endoplasmic reticulum (ER) (Browse and Somerville, 1991, Annu Rev Plant Physiol Plant Mol Biol 42:467). The FAD2 substrate 18:1 must be esterified on the sn-2 position of phosphatidylcholine (PC) (Miguel and Browse, 1992, J Biol Chem 267:1502; Okuley et al., 1994, Plant Cell 6:147), which is the major membrane phospholipid of plant cells. Not surprisingly, therefore, down-regulation of FAD2 (and FAD3) genes has become a preferred strategy for avoiding the need to hydrogenate vegetable oils and the concomitant production of undesirable trans fatty acids. For example, soybean has both seed-specific and constitutive FAD2 desaturases, so that gene silencing of the seed-specific isoform has allowed the production of high-oleate cultivars (>88% 18:1 in the oil) in which membrane unsaturation and plant performance are largely unaffected. Significantly, however, such FAD2 gene-silencing strategies are substantially limited because, for example, canola and other oilseed plants have only constitutive FAD2 enzymes. Therefore, in canola and other such constitutive FAD2 crops, silencing or down-regulation of FAD2 not only alters the fatty acid composition of the storage triacylglycerol (TAG) in seeds, but also of the cellular membranes, which severely compromises growth and yield of the plant. For example, the defective FAD2 in the Arabidopsis mutant fad2 alters fatty acid compositions of seeds as well as vegetable tissues, and severely compromises plant growth (Browse and Somerville, supra). FAD2 mutations and silencing that produce the highest 18:1 levels in the oil also reduce membrane unsaturation in vegetative and seed tissues, resulting in plants that germinate and grow poorly. As a result, only partial downregulation of FAD2 expression is possible, producing approximately 70-75% 18:1 in the oil of commercial cultivars such as Nexera/Natreon (Dow AgroSciences) and Clear Valley 75 (Cargill). Lu et al (2009, Proc Natl Acad Sci USA 106:18837) and WO2009/111587 describe the identification of phosphatidylcholine:diacylglycerol cholinephosphotransferase (PDCT) from Arabidopsis, which is endoced by the ROD1 gene, which is involved in the transfer of 18:1 into phosphatidylcholine for desaturation and also for the reverse transfer of 18:2 and 18:3 into the triacylglycerol synthesis pathway. The PDCT enzyme catalyzes transfer of 18:2 and 18:3 into the triacylglycerol synthesis pathway. Seeds of an Arabidopsis rod1 mutant have a decrease in 18:2 and 18:3 polyunsaturated fatty acids and a concomitant increase in 18:1 relative to wild-type, whereas there is no effect on the fatty acid compositions of leaf or root tissues. identified in Arabidopsis. WO2009/111587 further describes ROD1 homologs from Brassica napus, Brassica rapa, and Brassica oleracea. 
In order to use the ROD1 gene to increase 18:1 levels and reduce 18:2 and 18:3 levels in Brassica juncea, a need remains for knowing all ROD1 gene sequences and the functionality of the encoded proteins in the Brassica juncea genome. The isolation of mutant alleles corresponding to rod1 in Brassica juncea may be complicated by the amphidiploidy and the consequent functional redundancy of the corresponding genes.
Thus, the prior art is deficient in teaching the ROD1 gene sequences and the number of ROD1 genes in Brassica juncea, and which of the ROD1 genes encode a functional protein or need to be inactivated in order to increase the levels of 18:1 in Brassica juncea. As described hereinafter, this problem has been solved, allowing to modulate expression of PDCT with the aim to modulate the 18:1 levels in Brassica juncea, as will become apparent from the different embodiments and the claims.
USPTO Backgrounds
USPTO Backgrounds