Cloning and expression of a gibberellin 2β,3β-hydroxylase cDNA from pumpkin endosperm

Abstract

A cDNA expression library in λMOSElox derived from poly(A)+ RNA from pumpkin endosperm was screened immunologically with a polyclonal antibody raised against partially purified gibberellin (GA) 2β,3β-hydroxylase from pumpkin endosperm. A recombinant fusion protein encoded by a selected positive clone catalyzed 3β-hydroxylation of GA15, GA24, GA25, and GA17 and of GA12-aldehyde, GA12, GA9 and GA20, albeit less efficiently. The fusion protein also catalyzed 2β-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 pl 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 3β-hydroxylase enzyme activities were present in cell-free systems prepared from embryos of mature seeds and decreased after imbibition.