title: Isolation and expression of three gibberellin 20-oxidase cDNA clones from Arabidopsis.
abstract: Using degenerate oligonucleotide primers based on a pumpkin (Cucurbita maxima) gibberellin (GA) 20-oxidase sequence, six different fragments of dioxygenase genes were amplified by polymerase chain reaction from arabidopsis thaliana genomic DNA. One of these was used to isolate two different full-length cDNA clones, At2301 and At2353, from shoots of the GA-deficient Arabidopsis mutant ga1-2. A third, related clone, YAP169, was identified in the Database of Expressed Sequence Tags. The cDNA clones were expressed in Escherichia coli as fusion proteins, each of which oxidized GA12 at C-20 to GA15, GA24, and the C19 compound GA9, a precursor of bioactive GAs; the C20 tricarboxylic acid compound GA25 was formed as a minor product. The expression products also oxidized the 13-hydroxylated substrate GA53, but less effectively than GA12. The three cDNAs hybridized to mRNA species with tissue-specific patterns of accumulation, with At2301 being expressed in stems and inflorescences, At2353 in inflorescences and developing siliques, and YAP169 in siliques only. In the floral shoots of the ga1-2 mutant, transcript levels corresponding to each cDNA decreased dramatically after GA3 application, suggesting that GA biosynthesis may be controlled, at least in part, through down-regulation of the expression of the 20-oxidase genes.
authors.affiliation: IACR-Long Ashton Research Station, Department of Agricultural Sciences, University of Bristol, United Kingdom.
authors.forename: A L
authors.initials: AL
authors.lastname: Phillips
authors.forename: D A
authors.initials: DA
authors.lastname: Ward
authors.forename: S
authors.initials: S
authors.lastname: Uknes
authors.forename: N E
authors.initials: NE
authors.lastname: Appleford
authors.forename: T
authors.initials: T
authors.lastname: Lange
authors.forename: A K
authors.initials: AK
authors.lastname: Huttly
authors.forename: P
authors.initials: P
authors.lastname: Gaskin
authors.forename: J E
authors.initials: JE
authors.lastname: Graebe
authors.forename: P
authors.initials: P
authors.lastname: Hedden
mesh_terms: D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D018076:DNA, Complementary; D018744:DNA, Plant; D004926:Escherichia coli; D006899:Mixed Function Oxygenases / Q000235:genetics*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction
chemical_list: D017931:DNA Primers; D018076:DNA, Complementary; D018744:DNA, Plant; D006899:Mixed Function Oxygenases; C089210:gibberellin, 2-oxoglutarate-oxygen oxidoreductase (20-hydroxylating, oxidizing)