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. 1992 Dec;100(4):1751–1758. doi: 10.1104/pp.100.4.1751

Isolation and Characterization of Two Safflower Oleoyl-Acyl Carrier Protein Thioesterase cDNA Clones

Deborah S Knutzon 1,1, Janice L Bleibaum 1, Janet Nelsen 1, Jean C Kridl 1, Gregory A Thompson 1
PMCID: PMC1075860  PMID: 16653193

Abstract

Oleoyl-acyl carrier protein (18:1-ACP) thioesterase has been partially purified from developing safflower (Carthamus tinctorius) seeds. Protein species with molecular masses of 34 and 40 kD associated with thioesterase activity were identified and partially sequenced. Analysis of amino-terminal and internal cyanogen bromide peptide sequences revealed no differences in the primary structure of the two species. Amino acid sequence was used to design degenerate oligonucleotides for primers in a polymerase chain reaction (PCR) using safflower embryo cDNA as a template. A 380-base pair PCR product was used to isolate two classes of cDNA clones, designated 2-1 and 5-2, from the embryo cDNA library. Clone 2-1 encodes a 389-amino acid protein including a 60-amino acid transit peptide, and contains all of the protein sequence determined from the 34- and 40-kD proteins. Clone 5-2 encodes a 385-amino acid protein with 80% identity to that encoded by 2-1. Expression of the two safflower cDNA clones in Escherichia coli resulted in a 50- to 100-fold increase in the level of 18:1-ACP thioesterase activity. Both thioesterases are most active on 18:1-ACP; however, the enzyme encoded by 5-2 shows less discrimination against saturated 16- and 18-carbon acyl-ACP substrates.

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Selected References

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