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Cloning gibberellin dioxygenase genes from pumpkin endosperm by heterologous expression of enzyme activities in Escherichia coli
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| Zeitschriftentitel: | Proceedings of the National Academy of Sciences |
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| Personen und Körperschaften: | |
| In: | Proceedings of the National Academy of Sciences, 94, 1997, 12, S. 6553-6558 |
| Format: | E-Article |
| Sprache: | Englisch |
| veröffentlicht: |
Proceedings of the National Academy of Sciences
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| Schlagwörter: |
| Zusammenfassung: | <jats:p> 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 <jats:italic>Escherichia coli</jats:italic> . <jats:italic>In vitro</jats:italic> translation products of mRNA from endosperm of immature pumpkin seeds contained three GA dioxygenase activities, including 7-oxidase, 20-oxidase, and 3β-hydroxylase. A cDNA expression library was prepared from the endosperm mRNA in λMOS <jats:italic>Elox</jats:italic> . An aliquot of the amplified library was converted to plasmids <jats:italic>in vivo</jats:italic> and used for transformation of <jats:italic>E. coli</jats:italic> BL21(DE3), which thereafter expressed recombinant fusion proteins containing 7-oxidase, 20-oxidase, and 3β-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 <jats:italic>M</jats:italic> <jats:sub>r</jats:sub> of 35,712 and pI of 5.7. Recombinant GA 7-oxidase oxidizes GA <jats:sub>12</jats:sub> -aldehyde to GA <jats:sub>12</jats:sub> and GA <jats:sub>14</jats:sub> -aldehyde to GA <jats:sub>14</jats:sub> . Evidence was obtained for further metabolism of GA <jats:sub>12</jats:sub> by the 7-oxidase to four products, two of which are monohydroxylated GA <jats:sub>12</jats:sub> . 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. </jats:p> |
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| Umfang: | 6553-6558 |
| ISSN: |
1091-6490
0027-8424 |
| DOI: | 10.1073/pnas.94.12.6553 |