engleski

Insight into function and structure of gibberellin-20-oxidase from the Christmas rose (Helleborus niger L.)

The sepals of the Christmas rose (Helleborus niger L.) which are white or rose at anthesis, persist until the seeds are ripe and become intensely green which differs from reproductive development in commonly investigated model plants. This process is suppressed in unfertilized or depistillated flowers, but can be reinstated by treatment with gibberellins which indicates that endogenous gibberellins may play critical part in the metamorphosis of Christmas rose sepals. Previous studies showed that gibberellins GA1 and GA4 are major gibberellins with distinct concentration dynamics, while GA3 and GA7 are minor gibberellins in Christmas rose flower. Enzyme gibberellin-20-oxidase (GA 20-oxidase) is involved in biosynthesis of all four bioactive gibberellins found in Christmas rose. Partial GA 20-oxidase cDNA was generated from Christmas rose seeds by using degenerative primers, cloned into pRSET expression plasmid, and recombinant protein was produced in bacterial system. Whole cell proteins (containing GA 20-oxidase) were isolated and separated by SDS PAGE. Protein band corresponding to GA 20-oxidase was cut out of gel and used for custom service production of polyclonal antibodies by immunization of a rabbit. Obtained antibodies were purified and used for western blot analysis where a presence of GA 20-oxidase was screened in diverse developmental stages of Christmas rose flower. Bioinformatics analyses of GA 20-oxidase were mostly performed by using on line ExPasy programs (http://www.expasy.ch/).

Helleborus niger L.; gibberellin-20-oxidase

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