engleski

Detection of gibberellin-20-oxidase in Christmas rose (Helleborus niger L.) tissues in vitro

The gibberellins (GA) are plant hormones that participate in many aspects of plant development, including seed germination, flowering, fruit set, and shoot elongation. Such a complex network of plant development and growth processes in which GAs are involved reveals a highly organized regulation of GA levels via GA metabolism. The later steps in GA biosynthesis pathways are catalyzed by a group of enzymes called soluble 2-oxoglutarate- dependent dioxygenases (2ODDs). By acting on the GA skeleton at the 20, 3β or 2β positions ; 2ODDs can lead either to the formation of biologically active GAs or to their inactivation. The enzyme gibberellin-20-oxidase (GA 20-ox) catalyzes the final step in the formation of bioactive GAs by removing carbon- 20 through its oxidation, thereby enabling formation of the bioactive C19 structure. In this study, a partial GA 20-oxidase cDNA was generated from Christmas rose (Helleborus niger L.) seeds by using degenerative oligonucleotide primers, designed on the basis of nucleotide sequences from conserved regions of related enzymes in other plant species. Furthermore, GA 20-ox cDNA was cloned into a pRSET expression plasmid, and the recombinant GA 20-ox protein was produced in competent E. coli BL21(DE3)RIL+ strain. Inclusion bodies (containing GA 20- oxidase) were isolated and separated by SDS- PAGE. The protein band corresponding to GA 20- oxidase was cut out of the gel and used for custom service production of polyclonal antibodies by immunization of a rabbit. The obtained antibodies were purified and used for western blot analysis in order to screen tissues of diverse developmental stages of Christmas rose flower for the presence of GA 20-ox. Soluble proteins were extracted from Christmas rose seeds and such extracts were used to immunoprecipitate the GA 20-ox protein with the same polyclonal antibodies. The acquired protein bands which most likely corespond to our GA 20-ox were excised from the gels and prepared for further peptide sequencing by mass spectrometry. Bioinformatics analyses of GA 20-oxidase were mostly performed by using online ExPasy programs (http://www.expasy.ch/).

Christmas rose ; Gibberellin-20-oxidase ; Helleborus niger L.

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