Cloning and expression of a gibberellin 2 beta,3 beta-hydroxylase cDNA from pumpkin endosperm.

AUTOR(ES)

Lange, T

RESUMO

A cDNA expression library in lambda MOSEIox derived from poly(A)+ RNA from pumpkin endosperm was screened immunologically with a polyclonal antibody raised against partially purified gibberellin (GA) 2 beta,3 beta-hydroxylase from pumpkin endosperm. A recombinant fusion protein encoded by a selected positive clone catalyzed 3 beta-hydroxylation of GA15, GA24, GA25, and GA17 and of GA12-aldehyde, GA12, GA9, and GA20, albeit less efficiently. The fusion protein also catalyzed 2 beta-hydroxylation of the C20 GAS GA25, GA13, and, as identified putatively, GA28. The full-length clone contains an open reading frame of 1041 nucleotides encoding 346 amino acid residues with a predicted molecular weight of 38,992 and pI of 7.2. Transcript levels of this gene and of the previously cloned GA 7-oxidase and 20-oxidase genes from pumpkin endosperm rose until day 2 after the start of imbibition of the mature seeds, but only at one-two hundredth to one-six thousandth of the level found in the endosperm, as determined by quantitative reverse transcriptase-polymerase chain reaction. In contrast, GA 7-oxidase, 20-oxidase, and 3 beta-hydroxylase enzyme activities were present in cell-free systems prepared from embryos of mature seeds and decreased after imbibition.

Documentos Relacionados

• Cloning of cDNA encoding steroid 11 beta-hydroxylase (P450c11).
• Genes for catecholamine biosynthesis: cloning by expression and identification of the cDNA for rat dopamine beta-hydroxylase.
• cDNA cloning and nucleotide sequences of alpha 1 and alpha 2 thionins from hexaploid wheat endosperm.
• A cDNA encoding starch branching enzyme I from maize endosperm.
• Serum dopamine beta-hydroxylase activity in menstrual migraine.