Cloning and characterization of a cDNA encoding gibberellin 20-oxidase from rice (Oryza sativa) seedlings

Degenerate oligonucleotide primers based on the amino acid sequences of gibberellin (GA) 20-oxidases from pumpkin and Arabidopsis were used in nested polymerase chain reactions to generate an internal cDNA fragment from rice (Oryza saliva L.) seedlings. Using this fragment as a probe, a full-length cDNA (pOs20ox) was isolated from a cDNA library constructed from rice seedlings. The deduced amino acid sequence of Os20ox showed good identity (42-55%) with the GA 20-oxidases from pumpkin and Arabidopsis. Recombinant Os20ox protein, produced in Escherichia coli, catalyzed the conversion of GA(12) and GA(53) to GA(9) and GA(20), respectively. In contrast to the enzyme from immature pumpkin seeds, the recombinant rice GA 20-oxidase did not produce the tricarboxylic acids GA(25) and GA(17) from GA(12) and GA(53), respectively. The conversion rate of GA(53) was higher than that of GA(12), which is consistent with the relatively high abundance of 13-hydroxylated GAs in rice seedlings, Os20ox mRNA accumulated at higher levels in seedlings of two GA-deficient dwarf mutants than in normal plants. Treatment with uniconazole-P, an inhibitor of GA biosynthesis, increased abundance of the mRNA, while exogenously applied GA(3) decreased it. These results suggest that the expression of the Os20ox gene is regulated by the level of physiologically active GA.