Cloning and Expression of a Gibberellin 2P,3P=Hydroxylase cDNA from Pumpkin Endosperm

A cDNA expression library in hMOSElox derived from poly(A)+ RNA from pumpkin endosperm was screened immunologically with a polyclonal antibody raised against partially purified gibberellin (GA) 2P,3P-hydroxylase from pumpkin endosperm. A recombinant fusion protein encoded by a selected positive clone catalyzed 3 P-hydroxylation of GA15, GA24, GA25, and GA,, and of GAI2-aldehyde, GAI2, G 4 , and GAm, albeit less efficiently. The fusion protein also catalyzed 2P-hydroxylation of the C2, GAs GA25, GA,, and, as identified putatively, GAm 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 p l 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 leve1 found in the endosperm, as determined by quantitative reverse transcriptase-polymerase chain reaction. In contrast, GA 7-oxidase, 20-oxidase, and 3P-hydroxylase enzyme activities were present in cell-free systems prepared from embryos of mature seeds and decreased after imbibition.