Cloning and expression of a gibberellin 2 beta,3 beta-hydroxylase cDNA from pumpkin endosperm.
A cDNA expression library in lambda MOSEIox derived from poly(A)+ RNA from pumpkin endosperm was screened immunologically with a polyclonal antibody raised against partially purified gibberellin (GA) 2 beta,3 beta-hydroxylase from pumpkin endosperm. A recombinant fusion protein encoded by a selected positive clone catalyzed 3 beta-hydroxylation of GA15, GA24, GA25, and GA17 and of GA12-aldehyde, GA12, GA9, and GA20, albeit less efficiently. The fusion protein also catalyzed 2 beta-hydroxylation of the C20 GAS GA25, GA13, and, as identified putatively, GA28. The full-length clone contains an open reading frame of 1041 nucleotides encoding 346 amino acid residues with a predicted molecular weight of 38,992 and pI of 7.2. Transcript levels of this gene and of the previously cloned GA 7-oxidase and 20-oxidase genes from pumpkin endosperm rose until day 2 after the start of imbibition of the mature seeds, but only at one-two hundredth to one-six thousandth of the level found in the endosperm, as determined by quantitative reverse transcriptase-polymerase chain reaction. In contrast, GA 7-oxidase, 20-oxidase, and 3 beta-hydroxylase enzyme activities were present in cell-free systems prepared from embryos of mature seeds and decreased after imbibition.
Lange
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T
Albrecht-von-Haller-Institut für Pflanzenwissenschaften, Universität Göttingen, Germany. tlange@gwdg.de
Robatzek
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S
Frisse
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A
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics*; D018744:DNA, Plant / Q000235:genetics*; D015870:Gene Expression; D017343:Genes, Plant; D005875:Gibberellins / Q000378:metabolism; D006899:Mixed Function Oxygenases / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D010940:Plant Proteins; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012639:Seeds / Q000201:enzymology; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014675:Vegetables / Q000201:enzymology* / Q000235:genetics* / Q000254:growth & development
D017931:DNA Primers; D018076:DNA, Complementary; D018744:DNA, Plant; D005875:Gibberellins; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; D006899:Mixed Function Oxygenases; C106552:gibberellin 7-oxidase; C089210:gibberellin, 2-oxoglutarate-oxygen oxidoreductase (20-hydroxylating, oxidizing); C108187:gibberellin 2beta, 3beta-hydroxylase, Cucurbita maxima