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A Mutation in a 3-Keto-Acyl-ACP Synthase II Gene is Associated with Elevated Palmitic Acid Levels in Soybean Seeds

^a Crop Science Dep., North Carolina State Univ., Raleigh, NC 27695
^b USDA-ARS, North Carolina State Univ., Raleigh, NC 27695
^c USDA-ARS, Oilseeds and Bioscience Program, Beltsville, MD 20705. K. Aghoram, current address: Dep. of Biology and Health Science, Meredith College, Raleigh, NC 27607

View larger version (29K): [in a new window]   Fig. 1. Schematic representation of the steps of the fatty acid pathway in plants involved with saturated fatty acid biosynthesis. Key enzymes in the pathway are depicted in elliptical boxes. The condensation enzyme KAS II is part of the fatty acid synthase (FAS) complex. The mutant fap2 locus is believed to be the result a debilitating mutation in a KAS II gene. ACC, acetyl Co-A carboxylase; FAT-B, fatty acid thioesterase B; ACS, acyl-CoA synthase.

View larger version (90K): [in a new window]   Fig. 2. DNA gel blot analysis of GmKAS II genes in soybean. Genomic DNAs from Century (Fap2, Fap2) and C1727 (fap2, fap2) were digested with the restriction enzymes BamHI (B), HindIII (H), and XbaI (X). Blotted DNAs samples were hybridized to a 32P-labeled probe corresponding to a fragment of a soybean KAS II gene (AF244518).

View larger version (55K): [in a new window]   Fig. 3. Multiple sequence alignment of predicted soybean KAS II enzymes. AF244518 represents a full-length soybean KAS II sequence obtained from GenBank. GmKAS IIA and IIB represent the conceptual translations of the respective GmKASII gene sequences from cultivar Century. Dots depict amino acid residues identical among all three sequences. Shaded residues highlight dissimilar amino acids. An asterisk is located above the tryptophan residue (W) in the normal soybean sequences where the predicted protein derived from the mutant GmKAS IIA gene product from C1727 terminates due to the introduction of a premature stop codon.

View larger version (48K): [in a new window]   Fig. 4. CAPS analysis of normal versus mutant GmKAS IIA genes from soybean. (A) Partial genomic sequence of GmKAS IIA and GmKAS IIB genes from C1727 showing exon (shaded) and intron (nonshaded) sequences in the vicinity of the DdeI site used for CAPS analysis. The locations of primer sequences used to specifically amplify the GmKAS IIA isoform are shown with arrows. An asterisk is located above the polymorphic A residue that creates a unique DdeI site in the GmKAS IIA gene from C1727 that is a G residue in the corresponding gene from Century. All DdeI recognition sites (CTNAG) within the displayed sequences are underlined. (B) DdeI digestion of genomic DNAs amplified from Century (Fap2, Fap2), C1727 (fap2, fap2), and N02–4441 (fap2, fap2) using the primer sequences shown in (A). Arrows point to the 220- and 131-bp fragments diagnostic of germplasm possessing the mutant GmKAS IIA gene. M, 50-bp molecular weight ladder.