CLONING AND CHARACTERIZATION OF A CDNA-ENCODING GIBBERELLIN 20-OXIDASE FROM RICE (ORYZA-SATIVA) SEEDLINGS

Degenerate oligonucleotide primers based on the amino acid sequences o f gibberellin (GA) 20-oxidases from pumpkin and Arabidopsis were used in nested polymerase chain reactions to generate an internal cDNA frag ment from rice (Oryza saliva L.) seedlings. Using this fragment as a p robe, a full-length cDNA (pOs20ox) was isolated from a cDNA library co nstructed from rice seedlings. The deduced amino acid sequence of Os20 ox showed good identity (42-55%) with the GA 20-oxidases from pumpkin and Arabidopsis. Recombinant Os20ox protein, produced in Escherichia c oli, 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 conv ersion rate of GA(53) was higher than that of GA(12), which is consist ent with the relatively high abundance of 13-hydroxylated GAs in rice seedlings, Os20ox mRNA accumulated at higher levels in seedlings of tw o GA-deficient dwarf mutants than in normal plants. Treatment with uni conazole-P, an inhibitor of GA biosynthesis, increased abundance of th e mRNA, while exogenously applied GA(3) decreased it. These results su ggest that the expression of the Os20ox gene is regulated by the level of physiologically active GA.