CLONING AND EXPRESSION OF A GIBBERELLIN 2-BETA,3-BETA-HYDROXYLASE CDNA FROM PUMPKIN ENDOSPERM

A cDNA expression library in lambda MOSElox derived from poly(A)(+) RN A from pumpkin endosperm was screened immunologically with a polyclona l 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 o f CA(15), GA(24), GA(25), and GA(17) and of GA(12)-aldehyde, GA(12), G A(9), and GA(20), albeit less efficiently, The fusion protein also cat alyzed 2 beta-hydroxylation of the C-20 GAs GA(25), GA(13), and, as id entified putatively, GA(28). The full-length done contains an open rea ding 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 imbi bition of the mature seeds, but only at one-two hundredth to one-six t housandth of the level found in the endosperm, as determined by quanti tative reverse transcriptase-polymerase chain reaction, In contrast, G A 7-oxidase, 20-oxidase, and 3 beta-hydroxylase enzyme activities were present in cell-free systems prepared from embryos of mature seeds an d decreased after imbibition.