METHOD OF INCREASING ERUCIC ACID PRODUCTION BY HAIRPIN RNA FAD2 GENE SILENCING AND FAE GENE OVEREXPRESSION

Cross-reference to Related Application

This application claims the benefit of United States Provisional Patent Application USSN 60/996,739 filed December 3, 2007, the entire contents of which is herein incorporated by reference.

Field of the Invention

This invention relates generally to biotechnology and, more particularly to methods for producing bio-products in plants.

Background of the Invention

Modification of high erucic acid (HEA) germplasm of the Brassicaceae to increase the content of erucic acid (22:1δ13) in the seed oil for industrial niche markets (Jadhav et al., 2005) is an important goal in the industry. HEA cultivars are of high interest for industrial purposes because 22:1 is a valuable feedstock with more than 1000 potential or patented industrial applications (Scarth and Tang, 2006). Currently the major derivative of erucic acid is erucamide, which is used as a surface-active additive in coatings and in the production of plastic films as an anti-block or slip-promoting agent. Many other applications are foreseen for erucic acid and its hydrogenated derivative behenic acid, e.g. in lubricants, detergents, film processing agents and coatings, as well as in cosmetics and pharmaceuticals (Leonard, 1993; Derksen et al., 1995; Puyaubert et al., 2005; McVetty and Scarth, 2002; Mietkiewska et al., 2007). For many of these industrial uses, the economics are limited by the proportion of 22:1 in the seed oil. A Brassica cultivar containing erucic acid at levels approaching 80% would significantly reduce the cost of producing erucic acid and its derivatives and could meet the forecasted demand for erucic acid as a renewable, environmentally friendly industrial feedstock (Leonard, 1994; Taylor et al., 2002; Jadhav et al., 2005).

Over-expression of the Crambe abyssinica FAE gene in Brassica caήnata results in a substantial increase in the proportion of erucic acid in seeds compared to the wild type control (Mietkiewska et al., 2007). The synthesis of erucic acid in transgenic B. carinata plants was probably, in part, limited by the smaller microsomal pool of oleoyl- moieties (7-8%) available for elongation. As pointed out previously by Bao et al. (1998) and subsequently by Jadhav et al. (2005) the flux of oleic acid (18:1) through distinct

intermediate lipid pools before elongation might be a factor that limits the availability of 18:1 for elongation. The fatty acid desaturase (oleate desaturase), FAD2, is one of the crucial enzymes for the production of polyunsaturated fatty acids in plants (Okuley et al., 1994). By altering the level of FAD2 gene expression using antisense and cosuppression approaches, it was possible to increase the pool of 18:1 available for elongation to enhance production of erucic acid in B. caήnata seeds (Jadhav et al., 2005). However, the antisense and cosuppression strategies have variable and unpredictable effectiveness and require the production of large populations of transgenic plants to obtain a reasonable number of lines showing sufficient levels of target gene suppression (Liu et al., 2002).

The discovery that RNA interference in plants is mediated by sequence-specific degradation of dsRNA has led to the development of highly efficient methods of post transcriptional gene silencing (PTGS). Constructs specially designed to express dsRNA in plants in the form of self-complementary hairpin RNA (hpRNA) elicit a high degree and frequency of PTGS of endogenous genes (Smith et al., 2000; Stoutjesdijk et al., 2002). Such hpRNA constructs have great potential for genetic manipulation to improve crop traits (Wang et al., 2000).

B. carinata holds considerable promise as an alternative crop platform for industrial oil production and high-erucic oils in particular. S. carinata is easily transformed at very high efficiency (Babic et al., 1998), is highly disease (e.g. blackleg)-resistant, and is drought-resistant, amenable to growth in hotter, drier regions such as the brown soil areas of southern Saskatchewan. It also has negligible out-crossing to canola, and therefore poses little or no risk of contaminating oils destined for the food and feed chain.

There remains a need for transgenic B. carinata that produces high-erucic acid oils.

Summary of the Invention

There is provided a nucleic acid construct for increasing production of erucic acid in Brassica carinata plants, the construct comprising: an intron-spliced hairpin RNA nucleotide sequence for silencing fatty acid desaturase (FAD2) gene expression, the hairpin RNA nucleotide sequence comprising intron-interrupted inverted repeats of a nucleotide sequence cloned from the 3' untranslated region of a fatty acid desaturase (FAD2) gene of Brassica carinata; a heterologous nucleotide sequence from Crambe abyssinica coding for a fatty acid elongase (FAE); and, a seed-specific promoter.

There is further provided cells, plants and seeds of B. carinata comprising a construct of the present invention.

There is yet further provided a method of increasing erucic acid production in a Brassica carinata plant comprising transforming the plant with a construct of the present invention and growing the plant under conditions where the construct is expressed.

Thus, a partial 3'-UTR of the B. carinata FAD2 gene was used to prepare an intron-spliced hpRNA construct to silence the FAD2 gene and consequently, to increase the pool of oleic acid available for elongation. The increased pool of oleic acid contributes to a dramatic increase in the content of erucic acid in Brassica seeds when combined with heterologous C. abyssinica FAE expression. Any suitable intron, for example the intron from pyruvate orthophosphate dikinase (pdk) gene, may be used to separate the inverted repeats of the hairpin RNA.

Advantageously, as a result of the present invention, the level of erucic acid (22:1) in the triacylglycerols (TAGs) of cells, seeds and plants of the present invention is elevated by at least 10% by weight, based on weight of total TAGs, over the level of erucic acid in wild type cells, seeds and plants.

The seed-specific promoter allows for over-expression in seeds without affecting expression in other tissues. By way of illustration, preferred promoters used in over- expression of enzymes in seed tissue are the seed-specific napin promoter, and an ACP promoter as described in PCT International Publication WO 92/18634, published October 29, 1992, the disclosure of which is herein incorporated by reference. The promoter and termination regulatory regions will be functional in the host plant cell and may be heterologous (that is, not naturally occurring) or homologous (derived from the plant host species) to the plant cell and the gene. The seed-specific napin promoter is particularly preferred.

The termination regulatory region may be derived from the 3' region of the gene from which the promoter was obtained or from another gene. Suitable termination regions which may be used are well known in the art and include Agrobacterium tumefaciens nopaline synthase terminator (Nos T), A. tumefaciens mannopine synthase terminator (Mas T) and the CaMV 35S terminator (T35S). Particularly preferred termination regions for use herein include the Nos T termination region.

Preferably, a construct for use herein is comprised within a vector, most suitably an expression vector adapted for expression in an appropriate host (plant) cell. It will be

appreciated that any vector which is capable of producing a plant comprising the introduced nucleic acid sequence will be sufficient. Suitable vectors are well known to those skilled in the art and are described in general technical references such as Pouwels et al., (1986). Particularly suitable vectors include the pCR2.1 vector, the pRD400 vector and the Ti plasmid vector.

Transformation techniques for introducing the nucleic acid constructs into host cells are well known in the art and include such methods as micro-injection, using polyethylene glycol, electroporation, or high velocity ballistic penetration. A preferred method relies on /4groόactem//77-mediated transformation. After transformation of the plant cells or plant, those plant cells or plants into which the desired nucleic acid has been incorporated may be selected by such methods as antibiotic resistance, herbicide resistance, tolerance to amino-acid analogues or using phenotypic markers.

Various assays may be used to determine whether the plant cell shows an increase in gene expression, for example, Northern blotting or quantitative reverse transcriptase PCR (qRT-PCR). Whole transgenic plants may be regenerated from the transformed cell by conventional methods. Such plants produce seeds containing the genes for the introduced trait and can be grown to produce plants that will produce the selected phenotype.

Plants transformed with a construct of the instant invention may be grown. Seeds of the transgenic plants are harvested and the product of interest is extracted. The extracted product is used for subsequent incorporation into a composition, for example a pharmaceutical composition, a nutraceutical composition or a food composition.

The invention is susceptible to various modifications and alternative forms in addition to specific embodiments shown by way of example in the drawings and described in detail herein. Thus, the invention is not limited to the particular forms disclosed. Rather, the scope of the invention encompasses all modifications, equivalents, and alternatives falling within the following appended claims. Further features of the invention will be described or will become apparent in the course of the following detailed description.

Brief Description of the Drawings

In order that the invention may be more clearly understood, embodiments thereof will now be described in detail by way of example, with reference to the accompanying drawings, in which:

Fig. 1 is a schematic diagram (not to scale) of XD and XS constructs used to transform Brassica carinata plants. Both constructs, driven by napin promoter (Napin P), have an inverted repeat of a 142 bp fragment (3'-UTR) corresponding to the 3'-UTR of the B. carinata FAD2 gene (GenBank accession no: DQ250814) separated by the intron of pdk (Wesley et al. 2001). The XS construct also contains the coding region of the C. abyssinica FAE gene (CaFAE). The neomycin phosphotransferase gene (NPTW) is driven by the NOS promoter (NosP). The T-DNA left border (LB) and right border (RB) are shown. The positions of the restriction enzyme sites used for the cloning are as indicated.

Fig. 2 depicts proportion of unsaturated fatty acids in seed oils from nontransformed B. carinata (WT) and B. carinata T ₁ independent lines transformed with XD (A) and XS (B) constructs. The values are the average ± SD of three determinations.

Fig. 3 depicts correlation of FAD2 gene silencing activity expressed as an oleic acid desaturation proportion (ODP) value (gray bars) with erucic acid content (♦) in seed oils from nontransformed B. carinata (WT) and selected B. carinata T ₁ independent lines transformed with the XD and the XS constructs.

Fig. 4 depicts northern blot analysis of FAD2 gene expression in nontransformed β. carinata (WT) and in T1 transgenic seeds carrying the XD and XS constructs. (A) Total

RNA was isolated from mid developing seeds, blotted and probed with a 32P-labeled 0.5- kb fragment of the FAD2 gene. (B) The amount of RNA loaded per line was calibrated by the relative ethidium bromide staining of the ribosomal RNA bands.

Fig. 5 depicts fatty acid composition of individual lipids from mature seeds of wild type S. carinata (WT) and XS-18A transgenic line. The values are the average ± SD of three determinations.

Description of Preferred Embodiments

Materials and Methods:

Plant materials and growth conditions

Brassica carinata plants were grown under sterile conditions on MS medium (Murashige and Skoog, 1962) during transformation and tissue culture. Transgenic B. carinata plants were grown in the greenhouse at the Kristjanson Biotechnology Complex greenhouses, Saskatoon, SK, under natural light conditions supplemented with high- pressure sodium lamps with a 16 h photoperiod (16 h of light and 8 h of darkness) at 22 ⁰ C and a relative humidity of 25 to 30%.

Cloning of 3'-UTR of FAD2 gene

The ORF sequence of FAD2-gene (GenBank accession no. AF124360, SEQ ID

NO: 1) from B. carinata was used to design the forward primer 5'-

GTCTGCTACGGTCTCTACCG-3'. 3'-RACE was performed using the SMARTTM RACE kit (CLONTECH). A cDNA prepared from S. carinata developing seeds was used as a template for PCR amplification during 30 cycles of the following program: 94 ⁰ C for 30 sec,

58 ⁰ C for 30 sec, and 72 ⁰ C for 1 min. A 686-bp PCR product was cloned into the pCR2.1-

TOPO (Invitrogen) cloning vector and subsequently sequenced. Sequence comparison of the PCR product with the FAD2 ORF sequence showed the presence of 236-bp 3'-UTR. The 3'-UTR sequence was submitted to GenBank (accession no. DQ250814, SEQ ID

NO: 2).

Gene-silencing constructs

A 142 bp region of the FAD2 3'-UTR (SEQ ID NO: 3) was amplified by PCR with primers: 5'-ctcgag GGATGATGATGGTTTAAGA-3' (SEQ ID NO: 4) (lower case shows restriction site for Xhoϊ) and 5'-ggtaccCCATATCACATAATTTAAAGCC-3' (SEQ ID NO: 5) (lower case shows restriction site for Kpn\) and cloned in the sense orientation into Xho\ and Kpn\ sites of pKannibal resulting in pKannibal/A plasmid. Subsequently the 3' UTR was amplified with primers 5'-tctagaGGATGATGATGGTTAAGA-3' (SEQ ID NO: 6) (lower case shows restriction site for Xhoϊ) and 5'-aagcttCCATATCACATAATTTAAAGCC-3' (SEQ ID NO: 7) (lower case shows restriction site for Hind\\\) and then cloned in the antisense orientation in Hind\\\ and Xba\ sites of pKannibal/A giving pKannibal/A-B. The napin promoter (Josefsson et al., 1987) was ligated into pCR2.1 as an Xho\-Sac\ fragment. Then the Xho\-Xba\ cassette carrying intron-interrupted inverted repeats of the FAD2 3'-UTR was excised from pKANNIBAL/A-B and subsequently cloned into the respective sites of pCR2.1 vector (Invitrogen) behind the napin promoter. The resulting plasmid was named XC. A NOS terminator (Bevan, 1983) amplified by PCR with primers 5'-tctagaGATCGTTCAAACATTTGGCAA-3' (SEQ ID NO: 8) (lower case shows restriction site for Xba\) and 5'-ggtcgacCGATCTAGTAACATAGATGAC-3' (SEQ ID NO: 9) (lower case shows restriction site for Sa/I) and subsequently as Xba\-Sal\ fragment, was ligated with the Xba\-Sac\ fragment from the XC plasmid into the respective sites of pRD400 (CLONTECH). The resulting plasmid was named XD (Fig. 1).

Isolation of the Crambe abyssinica FAE gene was performed as described previously (Mietkiewska et al., 2007). A C. abyssinica ORF (SEQ ID NO: 10) was amplified by PCR with the primers: 5'-cccgggATGACGTTCCATTAACGTAAAG-3' (SEQ

ID NO: 11) (lower case restriction site for Sma\) and 5'-ggatccTTAGGACCGACCGTTTTGG-3' (SEQ ID NO: 12) (lower case restriction site for BamH\). The napin promoter was amplified with primers 5'-gaattcAAGCTTTCTTCATCGGTG-3' (SEQ ID NO: 13) (lower case restriction site for EcoRI) and 5'-cccgggGTCCGTGTATGTTTTTAATC-3' (SEQ ID NO: 14) (lower case restriction site for Smal). The NOS terminator was generated by PCR with the primers 5'-ggatccGATCGTTCAAACATTTGGCAA-3' (SEQ ID NO: 15) (lower case restriction site for BamH\) and 5'-gagctcCGATCTAGTAACATAGATGAC-3' (SEQ ID NO: 16) (lower case restriction site for Sacl). The napin promoter as an EcoR\-Sma\ fragment, the C. abyssinica FAE as an Sma\-BamH\ fragment and the Nos terminator as a BamH\-Sac\ fragment were ligated into the EcoRI-Sacl sites of pBluescript Il (SK+), resulting in plasmid ZB. Subsequently the EcoRI-Sacl cassette was excited from the ZB plasmid and cloned into the respective sites of the XD plasmid resulting in plasmid XS (Fig. 1).

The final binary vectors XD and XS were electroporated into Agrobacterium tumefaciens cells strain GV3101 containing helper plasmid pMP90 (Koncz and Schell, 1986). Plasmid integrity was verified by DNA sequencing following its re-isolation from A. tumefaciens and transformation into E. coli.

Plant transformation

Brassica carinata plants were transformed by the method of Babic et al. (1998). Transgenic plants were selected and analyzed as described by Mietkiewska et al. (2007).

Northern and Southern analysis

Total RNA from B. carinata plant material was isolated as described by Lindstom and Vodkin (1991). 20 micrograms of RNA was fractionated on a 1.4% (w/v) formaldehyde-agarose gel and the gels were then stained with ethidium bromide to ensure that all lanes had been loaded equally (Sambrook et al., 1989). The RNA was subsequently transferred to Hybond N+ membrane (Amersham Biosciences, Baie d ^' Urfe, Canada). A 0.5-kb probe containing the 3' part of FAD2 gene was generated by PCR using primers δ'-GTCTGCTACGGTCTCTACCG-S' (SEQ ID NO: 17) and δ'-TCATAACTTATTGTTGTACCAG-S' (SEQ ID NO: 18) and subsequently radioactively labeled with ³² P using a Random Primers Labeling kit (Invitrogen). Membranes were hybridized at 60 ⁰ C overnight. The filters were washed once in 1x SSPE, 0.1% SDS for 15 min and in 0.1x SSPE, 0.1% SDS for 5-10 min at the temperature of hybridization. The blots were exposed to X-OMAT-AR film (Kodak, Rochester, NY, USA).

Twenty micrograms of S. carinata genomic DNA was digested with the restriction enzyme EcoRI, and the resulting fragments were separated on a 0.9% (w/v) agarose gei and transferred to Hybond N+ nylon membrane via an alkali blotting protocol. For plants transformed with the XD construct, a 1.1-kb DNA fragment containing the napin promoter amplified by PCR using primers δ'-AAGCTTTCTTCATCGGTG-S' (SEQ ID NO: 19) and δ'-TCCGTGTATGTTTTTAATC-S' (SEQ ID NO: 20) was used as the probe. A 1.5-kb probe containing the coding sequence of C. abyssinica FAE was generated by PCR using primers δ'-ATGACGTCCATTAACGTAAAG-S' (SEQ ID NO: 21) and δ'-GGACCGACCGTTTTGGGC-S' (SEQ ID NO: 22) and was used for the analysis of plants transformed with the XS construct. The labeling and hydridization were as described above.

Lipid analyses

The total fatty acid content and acyl composition of B. carinata seed oils were determined by gas chromatography of the FAMEs with 17:0 FAME as an internal standard as described (Katavic et al., 2001 , Taylor et al., 2002, Mietkiewska et al., 2007). The lipid class separation was carried out according to the method of Christie (1982). Polar and neutral lipids species were separated by TLC on Silica Gel 60 H plates developed 4 cm in diethyl ether, air dried and then developed in hexane:diethyl etheπacetic acid (70:30:1 , v/v/v). Subsequently polar lipids were further developed in chloroform:methanol:acetic acid:water (25:10:3:1 , v/v/v). TLC regions containing lipid species were scraped and samples saponified with 2ml of 10% KOH in methanol at 80 ⁰ C for 2 h. Following isolation of the free fatty acids, FAMEs were produced using 3N methanolic HCI and extracted and analyzed by GC as described earlier (Taylor et al., 2002). Relative fatty acid compositions were calculated as the percentage that each fatty acid represented of the total fatty acids. An additional indirect method of assessing the cumulative effects of FAD2 activity during seed fatty acid synthesis through an oleic desaturation proportion (ODP) parameter was calculated as described by Stoutjesdijk et al. (2002).

Results and Discussion:

Fatty acid composition of preliminary transformants

Brassica carinata plants were transformed with two constructs: XD, targeted at the endogenous FAD2 gene utilizing intron-spliced hpRNA-mediated gene silencing, and XS targeted at silencing the FAD2 gene along with heterologous expression of the Crambe

abyssinica FAE gene (Fig. 1). Eleven T ₀ plants carrying the XD construct arising from 7 independent transgenic lines, were identified that were both resistant to kanamycin and PCR-positive for the presence of the transgene. Thirty six plants were transformed with the XS construct representing 20 independent lines (data not shown).

The fatty acid composition of T ₁ seeds from individual plants was determined for all transformants. The best independent transgenic lines are shown in Fig. 2A. Seed specific silencing of the FAD2 gene resulted in a significant reduction of the level of 18:2 from 19.0% in the wild type background to as low as 4.5 % in line XD-4D. The strong reduction of the level of 18:2 was directly correlated with a significant increase in the proportion of oleic acid up to 21.2% in the best transgenic line carrying the XD construct, compared to 5.7% in the wild type background. The increased pool of 18:1 was utilized by the endogenous microsomal elongation complex (FAE) and this led to the increase in the proportion of erucic acid to as high as 50.6% in line XD-5A, compared with 40.0% in WT seeds, a 26.5% proportional increase over wild-type levels. The high increase in erucic acid achieved in the current study through ihpRNA-mediated silencing of the FAD2 gene is considerably greater than that reported by Jadhav et al. (2005), where transformation with FAD2 sequence in an antisense orientation resulted in a net maximum of 17% increase in the proportion of erucic acid in the best line, compared to the WT B. carinata seeds.

In order to study whether the increased pool of oleic acid can contribute to the higher accumulation of erucic acid obtained by heterologous expression of C. abyssinica FAE, we also designed the XS construct (Fig. 1). As expected, the pronounced silencing of the oleoyl desaturase resulted in large reduction in linoleic acid and concomitant increases in the proportions of oleic acid (Fig. 2B). The level of 18:2 in transgenic lines carrying the XS transgene was as low as 3.2 % in XS-18A compared with 19.0% in WT. The increased pool of oleic acid in seeds of the XS transgenic plants was subsequently utilized by the heterologously-expressed C. abyssinica FAE which resulted in a net increase of the proportion of erucic acid in T1 segregating seeds of up to 41% compared to the wild type. Our current approach resulted in a higher accumulation of erucic acid than that formerly reported utilizing C. abyssinica FAE alone, which resulted in a 33% increase in 22:1 of T1 seed oils (Mietkiewska et al., 2007).

Effect of gene silencing on oleate desaturation levels in seed from preliminary transformants

Oleate desaturase is highly active in developing seeds of wild type β. carinata, with 85% of 18:1 being converted to 18:2 and 18:3, for an oleic acid desaturation proportion (ODP) value of 0.85 (Table 1). Many transgenic T1 plants carrying XD and XS constructs showed a considerable reduction in the ODP value, to as low as 0.39, indicating a profound (55%) down-regulation of oleate desaturation. Most transgenic plants have ODP values from 0.4-0.7 meaning that only 40-70% of oleic acid produced in developing seeds carrying the silencing constructs was converted to polyunsaturated 18- carbon fatty acids compared with 85% in nontransformed B. carinata seeds. As shown in Fig. 3 a positive correlation between the degree of FAD2 gene silencing (expressed as ODP value) and the content of erucic acid was observed. All transgenic plants analyzed in the current study showed some degree of FAD2 gene silencing (Table 1). An intron- spliced hpRNA-mediated (iHP) gene silencing technique used in the present study to modify the expression of the oleate desaturase in B. carinata seed resulted in a much higher efficacy of gene silencing than reported earlier antisense or cosuppression approach (Jadhav et al. 2005). In A. thaliana the highest efficiency of FAD2 gene silencing was achieved using an iHP construct utilizing a 120-bp fragment of FAD2 3'- UTR (Stoutjesdijk et al., 2002). The presence of the intron in an iHP construct may result in increased or more stable transcript levels than in the nonintron-containing hpRNA constructs (Tanaka et al., 1990; Stoutjesdijk et al., 2002).

Table 1

Frequency distribution of ODP value in seed of Brassica carinata (WT) and T1 seed of B. carinata transformed with XD and XS construct.

The stability and inheritance of ihpRNA-mediated gene silencing for lipid metabolism related genes has been shown (Stoutjesdijk et al., 2002: Liu et al., 2002); specifically, two published papers on the application of an ihpRNA approach for silencing FAD2 (Yang et al., 2006) and DGAT (Zhang et al., 2005) were based on the results from

TO plants. The high efficiency of gene silencing obtained through the use of ihpRNA- mediated PTGS makes this technique a very valuable contribution to practical trait modification in agricultural plants. This is particularly important for plants with low efficiency of transformation (Liu et al., 2002).

Effect of silencing construct on the FAD2 mRNA level

Based on high oleic acid levels, the 6 best lines were selected to examine the FAD2 mRNA level in transgenic seeds by northern analysis (Fig. 4). In all lines, the FAD2 mRNA level decreased compared to that of the wild type (WT). These results demonstrated a high correlation between the increase of the content of 18:1 in total seed lipids and the decrease of FAD2 transcript level. The highly elevated level of 18:1 in transgenic seeds resulted from lower expression of FAD2 mRNA in transgenic lines induced by PTG silencing. Similar trends have been reported from silencing FAD2 gene in tobacco and FAD2-1 in cotton (Yang et al., 2006; Liu et al., 2002).

Effect of silencing constructs on individual lipid classes in transgenic B. carinata seeds.

Individual lipids extracted from seed tissue were analyzed for fatty acid composition. In the seeds of XS-18A both polar and neutral lipids showed an increase in the level of 18:1 compared with WT seeds (Fig. 5). In particular, the XS-18A TAG content of 18:1 increased by 2.6 fold compared to the WT seeds at the expense of 18-carbon polyunsaturated fatty acids. Among polar lipids, the most pronounced increase of 18:1 was found in phosphatidylethanolamine (PE) and phosphatidylcholine (PC), as high as 7- fold and 4-fold, respectively. In PC from F>4D2-silenced seeds, the increase in the level of 18:1 was accompanied by strong reduction in the level of palmitic acid (16:0) of up to 37%. Similar changes in polar lipid class composition were reported for leaves and seeds of FAD2 silenced tobacco plants (Yang et al., 2006). These phenomena were also found in an Arabidopsis thaliana FAD2 mutant and in F/\D2-silenced Gossypium hirsutum plants (Miquel and Browse, 1992: Liu et al., 2002). As proposed by Yang et al. (2006) F/\D2-silencing may have effect on de novo 16:0 synthesis. The profound increase of 18:1 with the concomitant decrease of 18:2 may affect the fluidity of membranes, which could result in down-regulation of 16:0 content by a mechanism which is not yet understood. Nonetheless, the average reduction in total saturates from 6.8% in wild type B. carinata to 5.3% in present XS transgenic lines represents a significant improvement in the undesirable saturated fat content of the oil.

More importantly, the increase in erucic acid in TAGs, from 52.7% in WT, to 62.9% in the RNAi FAD2 + Crambe FAE transgenic line XS-18A (Fig. 5), constitutes the best result observed in transgenic manipulations of B. caήnata seed oil to date.

Informal Listing of Sequences:

SEQ ID NO: 1 - Brassica carinata FAD2 atgggtgcaggtggaagaatgcaggtttctccttctcccaagaagtccgaaaccgatacc atcaagcgtgttccctgcgaga cgcctcccttcacagtaggagagctcaagaaagccatcccaccgcactgtttcaaacgct ccatccctcgctctttctcctacc tcatctgggacatcatcgtagcctcctgcttctactacgtcgccaccacctacttccccc tcctccctcaccctctctcttacattgc ttggcctctctactgggcctgccaaggctgcgtcctaaccggcgtctgggtcatagccca cgaatgcggccaccacgctttca gcgactaccagtggctagacgacaccgtcggtctcatcttccattccttcctcctcgtcc cttacttctcctggaagtacagccat cgccgtcaccactccaacaccggttcgctcgagagagacgaggtgtttgtccccaagaaa aaatcagacatcaagtggta cggcaagtacctcaacaaccctctcggacgcaccgtgatgctaaccgtccagttcactct cggctggcccttgtacttggcctt caacgtctcgggcagaccttaccccgaggggttcgcctgccatttccacccgaacgctcc catctacaacgaccgtgaacg cctccagatatacgtctccgacgctggcatcctcgccgtctgctacggtctctaccgtta cgctgccgcgcagggagtggcctc gatggtctgcctctacggagttccgcttctgatagtcaacgcgttcctcgtcttgatcac ttacttgcagcacacgcatccttcgct gcctcactacgactcctctgagtgggattggttgaggggagcgttggccactgttgacag agactacggaatcttgaacaag gtcttccacaacatcacggacacgcacgtggcgcatcatctgttctccacgatgccgcat tatcacgcgatggaggctacga aggcgataaagccgattctgggagactattaccagttcgatgggacaccatgggttaagg cgatgtggagggaggcgaag gagtgtatctatgttgaaccggacaggcaaggtgagaagaaaggtgtgttctggtacaac aataagttatga SEQ ID NO: 2 - Brassica caήnata - 3'-UTR ggatgatgatggtttaagaacaaaaagatctattgtctctggtcgtctttgttttaagaa gctatgttttcatttcaataatctaaatta tccattttgttgtgttttctgacattttggctttaaattatgtgatatgggaagttttag tgtctaaatgtctttgtgtctgtattgttctcgaac tatcttaatgttgtggcgttagtgtaaaaaaaaaaaaaaaaaaaaaaaaa

SEQ ID NO: 3 - Brassica carinata - 3'-UTR 142 bp fragment ggatgatgatggtttaagaacaaaaagatctattgtctctggtcgtctttgttttaagaa gctatgttttcatttcaataatctaaatta tccattttgttgtgttttctgacattttggctttaaattatgtgatatggga

SEQ ID NO: 4 - Primer ctcgagggat gatgatggtt taaga

SEQ ID NO: 5 - Primer ggtaccccat atcacataat ttaaagcc

SEQ ID NO: 6 - Primer tctagaggat gatgatggtt aaga SEQ ID NO: 7 - Primer aagcttccat atcacataat ttaaagcc

SEQ ID NO: 8 - Primer tctagagatc gttcaaacat ttggcaa

SEQ ID NO: 9 - Primer ggtcgaccga tctagtaaca tagatgac SEQ ID NO: 10 - Crambe abyssinica FAE ORF atgacgtccattaacgtaaagctcctttaccattacgtcataaccaacctttttaacctc tgtttctttccgttaacggcgatcgtcgc cgggaaagcctctcggcttaccatagacgatcttcaccacttatattattcctatctcca acacaacgtcataaccatagctcca ctctttgcctttaccgttttcggttcgattctctacatcgtgacccggcccaaaccggtt tacctcgttgagtactcatgctaccttcc accaacgcagtgtagatcaagtatctccaaggtcatggatatattttatcaagtaagaaa agctgatccttttcgtaacgggac atgcgatgactcgtcctggcttgacttcttgaggaagattcaagaacgttcaggtctagg cgacgaaactcacggccccgag

ggactgcttcaggtccctccccggaagacttttgcggcggcgcgtgaagagacggag caagtaatcgtcggtgcgctgaaa aatctattcgagaacaccaaagttaaccctaaagatataggtatacttgtggtgaactca agcatgtttaatccaactccttcac tctcagcgatggtcgttaatactttcaagctccgaagtaacgtaagaagctttaaccttg gtggcatgggttgtagtgctggcgtt atagccattgatctggctaaggacttgttgcatgtccataaaaacacgtatgctcttgtg gtgagcacagagaacatcacttata acatttacgctggcgataatagatccatgatggtttcaaactgcttgttccgtgttggcg gggccgctattttgctctccaacaagc ctagagatcgaagacggtccaaatacgagctagttcacacggtccgaacacataccggag ctgatgacaagtctttccgat gcgtccaacaaggagacgatgagaacggcaaaaccggagtgagtttgtccaaggacataa ccgaggttgctggtcgaac ggttaagaaaaacatagcaacattgggtcctttgattcttcctttaagcgagaaacttct ttttttcgttaccttcatggccaagaaa cttttcaaagataaagttaagcattactatgtcccggacttcaagcttgctattgaccat ttttgtatacatgcgggaggcagagc cgtgatcgatgtgctagagaagaatttaggcctagcaccgatcgatgtagaggcatcaag atcaacgttacatagatttggta acacatcatctagctcaatatggtatgagttggcatacatagaggcaaaaggaaggatga agaaaggtaataaagtttggc agattgctttagggtcaggctttaagtgtaacagtgcggtttgggtagctttaagcaatg tcaaggcttcgacaaatagtccttgg gaacattgcatcgatagatacccggttaaaattgattctgattcagctaagtcagagact cgtgcccaaaacggtcggtccta a SEQ ID NO: 11 - Primer cccgggatga cgttccatta acgtaaag

SEQ ID NO: 12 - Primer ggatccttag gaccgaccgt tttgg

SEQ ID NO: 13 - Primer gaattcaagc tttcttcatc ggtg

SEQ ID NO: 14 - Primer cccggggtcc gtgtatgttt ttaatc

SEQ ID NO: 15 - Primer ggatccgatc gttcaaacat ttggcaa SEQ ID NO: 16 - Primer gagctccgat ctagtaacat agatgac

SEQ ID NO: 17 - Primer gtctgctacg gtctctaccg

SEQ ID NO: 18 - Primer tcataactta ttgttgtacc ag

SEQ ID NO: 19 - Primer aagctttctt catcggtg

SEQ ID NO: 20 - Primer tccgtgtatg tttttaatc SEQ ID NO: 21 - Primer atgacgtcca ttaacgtaaa g

SEQ ID NO: 22 - Primer ggaccgaccg ttttgggc

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Other advantages that are inherent to the structure are obvious to one skilled in the art. The embodiments are described herein illustratively and are not meant to limit the scope of the invention as claimed. Variations of the foregoing embodiments will be evident to a person of ordinary skill and are intended by the inventor to be encompassed by the following claims.