Cloning and characterization of a cDNA encoding a multifunctional gibberellin 20-oxidase from perennial ryegrass (Lolium perenne L.)

Using degenerated oligonucleotide primers derived from the gibberellin (GA) 20-oxidase sequence from pumpkin (Cucurbita maxima) and Arabidopsis thaliana, an internal fragment of 525 bp was amplified by nested polymerase chain reaction from cDNA of immature inflorescence of perennial ryegrass (Lolium perenne L.). The full-length ryegrass GA 20-oxidase genomic DNA sequence was isolated by genome walking. The deduced full-length cDNA clone (Lp20ox) of perennial ryegrass immature inflorescence was isolated by polymerase chain reaction. Sequence comparison reveals that the putative amino acid sequence shares 57.1% homology with pumpkin (C maxima) and 92.3% homology with wheat (Triticum aestivum). Expression of Lp20ox in Escherichia coli catalyzed the conversion of GA(12)- to GA(9) and GA(53)- to GA(20), indicating a highly specific GA 20-oxidase activity. Southern blot analysis suggests the presence of two copies of GA 20-oxidase in the ryegrass genome. Lp20ox mRNA accumulated in germinating seeds, expanding leaves, inflorescence, actively growing shoots and internodes, while it could not be detected in developing ovaries and immature seeds. (C) 2002 Elsevier Science Ireland Ltd. All rights reserved.