Cloning and Functional Analysis of Two Gibberellin 3β-hydroxylase Genes That are Differently Expressed during the Growth of Rice

We have cloned two gibberellin (GA) 3β-hydroxylase genes, OsGA3ox1 and OsGA3ox2, from rice by screening a genomic library with a DNA fragment obtained by PCR using degenerate primers. We have used full-scan GC-MS and Kovats retention indices to show function for the two encoded recombinant fusion proteins. Both proteins show 3β-hydroxylase activity for the steps GA20to$GA_1, \> GA_5$to$GA_3, \>GA_{44}$to GA38and GA9to GA4. In addition, indirect evidence suggests that the OsGA3ox1 protein also has 2,3-desaturase activity, which catalyzes the steps GA9to 2,3-dehydro-GA9and GA20to GA5(2,3-dehydro GA20), and 2β-hydroxylase activity, which catalyzes the steps GA1to GA8and GA4to GA34. Molecular and linkage analysis maps the OsGA3ox1 gene to the distal end of the short arm of chromosome 5; the OsGA3ox2 gene maps to the distal end of the short arm of chromosome 1 that corresponds to the D18 locus. The association of the OsGA3ox2 gene with the d18 locus is confirmed by sequence and complementation analysis of three d18 alleles. Complementation of the d18-AD allele with the OxGA3ox2 gene results in transgenic plants with a normal phenotype. Although both genes show transient expression, the highest level for OsGA3ox1 is from unopened flower. The highest level for OsGA3ox2 is from elongating leaves.