title: Characterization of an oleate 12-desaturase from Physaria fendleri and identification of 5'UTR introns in divergent FAD2 family genes.
abstract: Mining of an EST sequence collection representing genes expressed during seed development in Physaria fendleri identified abundant sequences encoding apparent homologues of the Arabidopsis oleate 12-desaturase (AtFAD2 At3g12120). Of the 62 sequenced clones, 59 were identified as encoding the previously characterized bifunctional oleate 12-hydroxylase/desaturase (LFAH12/PfFAH12). The remaining 3 clones encoded a second FAD2 homologue. Isolation of a full length ORF and heterologous expression in yeast revealed that this sequence, designated PfFAD2, is the first full length sequence from any Physaria species that encodes an oleate 12-desaturase. PfFAD2 was expressed in both leaf and developing seed with activity on palmitate (16:1(Δ9)) and oleate (18:1(Δ9)). Sequence comparison revealed that PfFAD2 shares 93% amino acid identity with Arabidopsis FAD2 and only 84% identity with PfFAH12. By comparison of EST and genomic sequences it was revealed that the PfFAD2 gene encodes a transcript with a single intron of 1120 bp in the 5'-untranslated region (5'UTR). A short intron, 81 bp in length, was also discovered in the 5'UTR of the PfFAH12 gene, 16 bp upstream of the translation initiation codon. In silico examination of FAD2 like genes from the genome of castor (Ricinus communis) identified putative 5'UTR introns in genes encoding the castor oleate 12-desaturase (RcFAD2) and oleate 12-hydroxylase (CFAH12). By sequencing of genomic DNA the presence of single 5'UTR introns in each gene, and the size of these introns, was confirmed. These findings suggest that 5'UTR introns may be a characteristic feature of FAD2 genes and also of divergent FAD2 genes encoding fatty acid modifying enzymes, and that the selection pressure maintaining these introns is very different.
authors.affiliation: National Research Council of Canada, 110 Gymnasium Place, Saskatoon, SK S7N 0W9, Canada.
authors.forename: Sharla
authors.initials: S
authors.lastname: Lozinsky
authors.affiliation: National Research Council of Canada, 110 Gymnasium Place, Saskatoon, SK S7N 0W9, Canada.
authors.forename: Hui
authors.initials: H
authors.lastname: Yang
authors.affiliation: National Research Council of Canada, 110 Gymnasium Place, Saskatoon, SK S7N 0W9, Canada.
authors.forename: Li
authors.initials: L
authors.lastname: Forseille
authors.affiliation: National Research Council of Canada, 110 Gymnasium Place, Saskatoon, SK S7N 0W9, Canada.
authors.forename: Gillian R
authors.initials: GR
authors.lastname: Cook
authors.affiliation: National Research Council of Canada, 110 Gymnasium Place, Saskatoon, SK S7N 0W9, Canada.
authors.forename: Irving
authors.initials: I
authors.lastname: Ramirez-Erosa
authors.affiliation: National Research Council of Canada, 110 Gymnasium Place, Saskatoon, SK S7N 0W9, Canada. Electronic address: Mark.Smith@nrc-cnrc.gc.ca.
authors.forename: Mark A
authors.initials: MA
authors.lastname: Smith
mesh_terms: D020121:5' Untranslated Regions; D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000235:genetics / Q000378:metabolism; D019607:Brassicaceae / Q000235:genetics* / Q000378:metabolism; D018387:Codon, Initiator; D020224:Expressed Sequence Tags; D044943:Fatty Acid Desaturases / Q000235:genetics* / Q000378:metabolism; D017343:Genes, Plant; D007438:Introns; D006899:Mixed Function Oxygenases / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D019301:Oleic Acid / Q000235:genetics* / Q000378:metabolism; D044925:Oxidoreductases Acting on CH-CH Group Donors / Q000235:genetics / Q000378:metabolism; D018515:Plant Leaves / Q000378:metabolism; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism; D012278:Ricinus / Q000235:genetics / Q000378:metabolism; D012639:Seeds / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017385:Sequence Homology
chemical_list: D020121:5' Untranslated Regions; D018387:Codon, Initiator; D010940:Plant Proteins; D019301:Oleic Acid; D006899:Mixed Function Oxygenases; C094812:phosphatidylcholine 12-monooxygenase; D044943:Fatty Acid Desaturases; C021904:Phosphatidylcholine desaturase; D044925:Oxidoreductases Acting on CH-CH Group Donors
keywords: 5′UTR intron; EST; FAD2; FAMES; Fatty acid desaturase; Fatty acid hydroxylase; HFA; Lesquerella; ORF; Physaria fendleri; expressed sequence tag; fatty acid desaturase 2; fatty acid methyl esters; hydroxy fatty acid; open reading frame