Cloning gibberellin dioxygenase genes from pumpkin endosperm by heterologous expression of enzyme activities in Escherichia coli

Abstract

Gibberellin (GA) plant hormones are biosynthesized via complex pathways, the final steps of which are catalyzed by 2-oxoglutarate-dependent dioxygenases. Here, the cloning of two such enzymes, the GA 7-oxidase and the GA 20-oxidase, is reported using a novel approach, namely, by screening for GA dioxygenase activities expressed as T7 gene 10 fusion proteins in recombinant Escherichia coli. In vitro translation products of mRNA from endosperm of immature pumpkin seeds contained three GA dioxygenase activities, including 7-oxidase, 20-oxidase, and 3␤-hydroxylase. A cDNA expression library was prepared from the endosperm mRNA in MOSElox. An aliquot of the amplified library was converted to plasmids in vivo and used for transformation of E. coli BL21(DE3), which thereafter expressed recombinant fusion proteins containing 7-oxidase, 20-oxidase, and 3␤-hydroxylase activities. By screening for specific GA dioxygenase expression, clones harboring 7-oxidase and 20-oxidase cDNA were isolated. The ORF of the 7-oxidase cDNA is 945 bp long, encoding for 314 amino acid residues with a calculated M r of 35,712 and pI of 5.7. Recombinant GA 7-oxidase oxidizes GA 12 -aldehyde to GA 12 and GA 14 -aldehyde to GA 14 . Evidence was obtained for further metabolism of GA 12 by the 7-oxidase to four products, two of which are monohydroxylated GA 12 . The ORF of the 20-oxidase is-apart from seven changes, resulting in four amino acid substitutions-identical to the 20-oxidase cDNA previously cloned from pumpkin cotyledon mRNA; both 20-oxidases have the same catalytic properties.

Cloning and Molecular Analyses of a Gibberellin 20-Oxidase Gene Expressed Specifically in Developing Seeds of Watermelon

“…The PCR product was cloned in the pGEM-T Easy vector (Promega), and its sequence was determined to confirm the apparent GA 20-oxidase identity. Sequence comparison against genes in the database by the BLAST search program showed that its deduced amino acid sequence is highly homologous to the GA 20-oxidase from various plant species (Lange et al, 1994;Phillips et al, 1995;Xu et al, 1995;Garcia-Martinetz et al, 1997;Lange, 1997;MacMillan et al, 1997).…”

“…1). The watermelon protein shares 76% and 63% identities with the GA 20-oxidases of M. macrocarpus (MacMillan et al, 1997) and pumpkin (Lange, 1997), respectively. The protein also shares Ͼ50% identity with the GA 20-oxidases of pea (Garcia-Martinez et al, 1997), bean (Garcia-Martinez et al, 1997), Arabidopsis (Phillips et al, 1995), spinach (Wu et al, 1996), and lettuce (Toyomasu et al, 1998).…”

“…The 3␤-hydroxylases catalyze 3␤-hydroxylation of the C 19 skeleton, resulting in the formation of GAs that are biologically active. Genomic and cDNA clones encoding GA 20-oxidase and 3␤-hydroxylase have been isolated from several plant species, and their enzymatic properties have been identified with recombinant enzymes produced in Escherichia coli (Lange et al, 1994;Chiang et al, 1995;Phillips et al, 1995;Xu et al, 1995;Wu et al, 1996;Garcia-Martinez et al, 1997;Hedden and Kamiya, 1997;Lange, 1997;Lester et al, 1997;MacMillan et al, 1997;Toyomasu et al, 1998).…”

“…However, little is known about the roles of GAs and the exact locations of synthesis in developing seeds. It has recently been suggested that GAs are involved in the early and late stages of seed development in some plant species (Eeuwens and Schwabe et al, 1975;Graebe, 1987;Phillips et al, 1995;Lange, 1997;MacMillan et al, 1997;Swain et al, 1997). Several GA-deficient mutants that have altered seed development have been reported in the pea plant.…”

Gibberellin Is Essentially Required for Carrot (Daucus carotaL.) Somatic Embryogenesis: Dynamic Regulation of Gibberellin 3-Oxidase Gene Expressions

“…[14][15][16][17][18][19][20][21][22][23] Some evidence has been accumulating that suggests GA plays in the formation and development of zygotic embryos. For instance, relatively high levels of GAs were observed in an early stage of zygotic embryos in stenospermocarpic grape 24) and maize.…”