Molecular cloning and functional expression of the gene for a cotton Δ-12 fatty acid desaturase (FAD2)

• Published in: Biochimica et biophysica acta 2001-12, Vol.1522 (2), p.122-129
• Main Authors: Pirtle, Irma L., Kongcharoensuntorn, Wisatre, Nampaisansuk, Mongkol, Knesek, John E., Chapman, Kent D., Pirtle, Robert M.
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Base Sequence; Cloning, Molecular; Cotton genomic DNA; DNA - chemistry; Fatty acid desaturase 2; Fatty Acid Desaturases - biosynthesis; Fatty Acid Desaturases - chemistry; Fatty Acid Desaturases - genetics; Gene Expression Regulation; Gossypium - enzymology; Gossypium - genetics; Gossypium hirsutum; Integral membrane protein; Linoleic Acid - metabolism; Membrane Lipids - metabolism; Membrane Proteins - genetics; Molecular Sequence Data; Plant Proteins - genetics; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae - metabolism; Sequence Alignment; Unsaturated fatty acid; Δ-12 fatty acid desaturase 2 gene
• ISSN: 0167-4781; 0006-3002; 1879-2634
• DOI: 10.1016/S0167-4781(01)00312-8

Two overlapping genomic clones spanning 16.5 kb of cotton DNA were found to encompass a Δ-12 fatty acid desaturase ( FAD2-3) gene. A partial FAD2-3 cDNA clone was also analyzed. The FAD2-3 gene has one large intron of 2967 bp entirely within its 5′-untranslated region, only 12 bp upstream from the ATG initiation codon. Several potential promoter elements, including several light-responsive motifs, occur in the 5′-flanking region. The continuous FAD2-3 coding region is 1155 bp and would encode a protein of 384 amino acids. The polypeptide has four putative membrane-spanning helices, indicative of an integral membrane protein, and is most likely localized in the endoplasmic reticulum. Yeast cells transformed with a plasmid construct containing the cotton FAD2-3 coding region accumulate an appreciable amount of linoleic acid (18:2), not normally present in wild-type yeast cells, indicating that the gene encodes a functional FAD2 enzyme.