Cloning and functional analysis of two gibberellin 3 beta-hydroxylase genes that are differently expressed during the growth of rice

We have cloned two gibberellin (CA) 3 beta -hydroxylase genes, OsCA3ox1 and OsGA3ox2, from rice by screening a genomic library with a DNA fragment obt ained by PCR using degenerate primers. We have used full-scan CC-MS and Kov ats retention indices to show function for the two encoded recombinant fusi on proteins. Both proteins show 3 beta -hydroxylase activity for the steps GA(20) to GA(1), GA(5) to GA(3), GA(44) to GA(38) and GA(9) to GA(4). In ad dition, indirect evidence suggests that the OsGA3ox1 protein also has 2,3-d esaturase activity, which catalyzes the steps GA(9) to 2,3-dehydro-GA(9) an d GA(20) to GA(5) (2,3-dehydro GA(20)), and 2 beta -hydroxylase activity, w hich catalyzes the steps GA(1) to GA(8) and GA(4) to GA3(4) Molecular and l inkage analysis maps the OsGA-ox1 gene to the distal end of the short arm o f chromosome 5; the OsGA3ox2 gene maps to the distal end of the short arm o f chromosome 1 that corresponds to the D18 locus. The association of the Os GA3ox2 gene with the d18 locus is confirmed by sequence and complementation analysis of three d18 alleles. Complementation of the d18-AD allele with t he OxGA3ox2 gene results in transgenic plants with a normal phenotype. Alth ough both genes show transient expression, the highest level for OsGA3ox1 i s from unopened flower. The highest level for OsGA3ox2 is from elongating l eaves.