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Improving Erucic Acid Content in Rapeseed through Biotechnology

What Can the Arabidopsis FAE1 and the Yeast SLC1-1 Genes Contribute?¹,^,2

^a Saskatchewan Wheat Pool Agricultural Research and Development, 201-407 Downey Road, Saskatoon, SK, S7N 4L8, Canada
^b National Research Council of Canada, Plant Biotechnology Institute, 110 Gymnasium Place, Saskatoon, SK, S7N 0W9, Canada

View larger version (16K): [in a new window]   Fig. 1. The expression cassette comprising of napin promoter region (NAP), coding region for Arabidopsis microsomal fatty acid elongase 1 gene (FAE1) and nopaline-synthase terminator (NOS). Restriction sites XbaI and SstI were used to replace the GUS gene in vector pNap:GUS/NGKM and develop binary vector pNap: FAE1/NGKM (Millar and Kunst, 1997); NPTII: neomycin–phosphotransferase, RB: right border.

View larger version (69K): [in a new window]   Fig. 2. PCR amplification of the partial expression cassette NAP/FAE1/NOS using oligonucleotide primer NN-3 (5'-TTTCTTCGCCACTTGTCACTCC-3') which was designed according to the promoter region of the napin gene (position 948-969) and primer NN-4 (5'CGCGCTATATTTTGTTTTCTA-3') which was designed according to the nopaline-synthase 3' UTR sequence (position 1753–1773). The total size of the PCR product is approx. 2.0 kb (0.197 kb of napin promoter region + 1.608-kb FAE1 coding region + 0.204 kb of nopaline-synthase 3' UTR region); WS: Westar transgenic lines; WS-WT: Westar wild-type control line; C-: negative PCR control without DNA; H: Hero transgenic lines; H-WT: Hero wild-type control line; C+: positive PCR control—binary vector pNap:FAE1/NGKM.

View larger version (38K): [in a new window]   Fig. 3. The accumulation of erucic acid (22:1), eicosenoic acid (20:1), and very long chain fatty acids (VLCFAs) in T2 mature seeds of non-tranformed wild-type control (W-ntCon) lines and Westar FAE1 transgenic lines. Fatty acid levels are shown as g kg-1 of total extractable fatty acids. Each bar represents the average of ten samples ± SD, with single-seed being analyzed in each sample.

View larger version (30K): [in a new window]   Fig. 4. Elongase assays of seed homogenates from control and FAE1 transgenic lines. Shown are traces from reverse-phase HPLC analyses of radiolabeled fatty acid methyl esters. Developing seed extracts from (A) wild-type Westar control (WS-WT); (B)T2 Westar FAE1 transgenic line WS-2-10; (C) Hero wild-type control (H-WT) and (D) T2 Hero FAE1 transgenic line H-10-2, were incubated with [14C]18:1-CoA in the presence of malonyl-CoA, and elongase assays conducted as described by Taylor et al. (1992). Reaction mixtures were normalized with respect to the quantity of homogenate protein added, and therefore the data are presented as relative 14C radioactivity in FAME products from each reaction. Radiolabeled FAMEs were identified by co-chromatography with external standards.

View larger version (44K): [in a new window]   Fig. 5. The accumulation of eicosenoic acid, erucic acid (22:1), and very long chain fatty acids (VLCFAs) in T2 mature seeds of Hero non-transformed wild-type controls (H-ntCon) and Hero FAE1 transgenic lines. Fatty acid levels are shown as g kg-1 of total extractable fatty acids. Each bar represents the average of 10 samples ± SD, with single-seed being analyzed in each sample.

View larger version (43K): [in a new window]   Fig. 6. Erucic acid yield (g/plot) from field trials of B. napus cv. Hero non-transformed wild-type controls (H-ntCon) and of SLC1-1 T5 transgenic lines. Values are the averages of samples from two to five plots of each line ± standard error of the mean.

View larger version (40K): [in a new window]   Fig. 7. "Nearest neighbor" analysis of seed yield (g/plot) from B. napus cv. Hero non-transformed wild-type control (Con) and SLC1-1 T5 transgenic line 5-4-8 in field trials at Rosthern, SK, summer of 1999. ## > indicates the relative plot position within the test block, with plots numbered 1-72, consecutively.