Naslov
Characterization of the gibberellin 20-oxidase expression patterns in different developmental stages of Christmas rose (Helleborus niger L.) flower and seeds
(Characterization of the gibberellin 20-oxidase expression patterns in different developmental)

Autori
Brcko, Ana ; Mihaljević, Snježana ; Salopek-Sondi, Branka

Vrsta, podvrsta i kategorija rada
Sažeci sa skupova, sažetak, znanstveni

Izvornik
1st International Symposium of Biotech students, Book of Abstracts / - Zagreb : Student Association of Biotechnology-Helix, 2009, 21-22

Skup
1st International Symposium of Biotech students

Mjesto i datum
Zagreb, Hrvatska, 24.09.2009. - 26.09.2009

Vrsta sudjelovanja
Poster

Vrsta recenzije
Domaća recenzija

Ključne riječi
Helleborus niger L. ; GA 20-oxidase ; cDNA construction ; bioinformatic analysis

Sažetak
The Christmas rose (Helleborus niger L.) has emerged as a model plant with great potential to clarify the regulation of production and consumption of assimilates, because its perianth, which is white or rose at anthesis, persist until the seeds are ripe and becomes intensely green. The greening process of the perianth (formed in this species by sepals) differs from reproductive development in commonly investigated model plants. Furthermore, investigation of endogenous hormones, as potential signaling molecules between seeds and sepals in Helleborus, indicated gibberellins GA1 and GA4 as major bioactive gibberellins in sepals and fruit. Unfertilized, depistillated, and deseeded flowers become less green than the seed-bearing control ; whereas the external application of GA1 and GA4 can reinstate the chlorophyll accumulation. In this study, we aimed at characterizing a gibberelin 20-oxidase (HnGA20ox), which is one of the soluble 2- oxoglutarate-dependent dioxygenases involved in final steps of the formation of biologically active gibberellins. We have generated a partial HnGA20ox cDNA from Christmas rose seeds using degenerative primers. Primers were created by comparing nucleotide sequences of the conserved regions of related enzymes in other plant species. The cDNA of HnGA20ox was cloned into a pRSET expression plasmid, and the recombinant HnGA20ox protein was produced in competent E. coli BL21(DE3)RIL+ strain. The recombinant protein of around 24 kDa was successfully expressed in E. coli. Polyclonal antibodies were raised against recombinant HnGA20ox in a rabbit. Western blot analysis of the expression of HnGA20ox protein in different developmental stages of Cristmas rose flower showed the positions of potential GA 20- oxidases. The same antibodies also immuno- precipitated HnGA20ox (around 45 kDa) isolated from almost ripe Christmas rose seeds. Bioinformatics analysis of HnGA20ox was mostly performed using online ExPasy programs (http://www.expasy.ch/). Alignment of the partial HnGA20ox protein sequence from Christmas rose with GA 20-oxidases from other plant species was performed by T-Coffee and displayed in the GeneDoc program. Partial HnGA20ox, analyzed by ProtParam, is a 24 kDa protein containing 212 amino acids and its theoretical pI is 6, 81. The protein is classified as stable according to the Prot Param tool for primary structure analysis. The tertiary structure was predicted with CPHmodels-2.0 and visualized by PyMol. The template used for generating this model was the file 1GP5.pdb for anthocyanidin synthase from Arabidopsis thaliana because so far there is no known tertiary structure of any GA 20-oxidase. Amino acid identity for these two proteins is 28, 4%.

Izvorni jezik
Engleski

Znanstvena područja
Biologija

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