Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
Comparative Study
. 1997 Jun 10;94(12):6553-8.
doi: 10.1073/pnas.94.12.6553.

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

T Lange  1
Affiliations
Comparative Study

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

T Lange. Proc Natl Acad Sci U S A. .

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 3beta-hydroxylase. A cDNA expression library was prepared from the endosperm mRNA in lambdaMOSElox. 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 3beta-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 Mr of 35,712 and pI of 5.7. Recombinant GA 7-oxidase oxidizes GA12-aldehyde to GA12 and GA14-aldehyde to GA14. Evidence was obtained for further metabolism of GA12 by the 7-oxidase to four products, two of which are monohydroxylated GA12. 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.

PubMed Disclaimer

Figures

Figure 1
Figure 1
GA biosynthetic pathways to C20-tricarboxylic acids (GA25, GA13, GA43) and C19-GAs (GA9, GA4) as found in pumpkin endosperm (2, 3) and reactions shown to be catalyzed by recombinant 7-oxidase (A) and 20-oxidase (B).
Figure 2
Figure 2
GA 7-oxidase, 20-oxidase, and 3β-hydroxylase activity in E. coli BL21(DE) harboring pMOSElox cDNA expression library. [14C]GA12-aldehyde (a, c) and [14C]GA12 (b, d) were incubated with lysates prepared from bacteria with pMOSElox plasmids without insert (a, b) or with 59,000 (c) or 8,400 (d) clones of the pMOSElox cDNA library derived from pumpkin endosperm poly(A)+ RNA. On the basis of HPLC retention times peaks correspond to GA12-aldehyde (I), GA12 (II), GA15 (III), and GA37 (IV).
Figure 3
Figure 3
Alignment of derived GA 7-oxidase (Cm7ox) and 20-oxidase (Cm20ox) amino acid sequences from pumpkin endosperm with GA 20-oxidase (At2353) (9) and putative 3β-hydroxylase sequence (At3h) (16) from Arabidopsis. Identical residues are boxed in black; similar residues are shaded in gray.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Crozier A, editor. The Biochemistry and Physiology of Gibberellins. Vol. 2. New York: Praeger; 1983.
    1. Graebe J E. Annu Rev Plant Physiol. 1987;38:419–465.
    1. Lange T, Hedden P, Graebe J E. Planta. 1993;189:340–349. - PubMed
    1. Lange T, Hedden P, Graebe J E. Planta. 1993;189:350–358. - PubMed
    1. Lange T, Schweimer A, Ward D A, Hedden P, Graebe J E. Planta. 1994;195:98–107.

Publication types

MeSH terms

Substances

Associated data