Štúdium MMK3 proteínu v Lucerne siatej a jeho ortológu MPK13 v arábkovke Thalovej

Janka LOKAJOVÁ

Bachelor's thesis

Study of the MMK3 protein in alfalfa and its MPK13 ortholog in Mouse-ear thale-cress

Abstract:

Pojem mitogen aktivované proteín kinázy zahŕňa signálne proteíny zodpovedné za prenos extracelulárnych signálov a reguláciu bunkových procesov. Ich dôležitosť vyplýva zo sesilnej povahy rastlinných organizmov, ktoré sú vystavované nepriaznivým podmienkam okolia a práve MAP kinázová kaskáda predstavuje jeden zo spôsobov obrany. V tejto bakalárskej práci boli pripravené konštrukty MMK3 proteínu vo fúzii s fluorescenčným proteínom. Konštrukty boli validované pomocou metódy tranzientnej transformácie listov Nicotiana benthamiana a metódy Western-blot. Následne boli použité pre stabilnú transformáciu v Medicago sativa (RSY), a tiež heterologicky v Arabidopsis thaliana (Col 0, mpk13). Transformované rastliny boli overené PCR genotypizáciou prípadne Western blot analýzou. Taktiež boli vykonané fenotypové analýzy mutantných línii A. thaliana mpk13, ktoré poukázali na rozdiely vo vývoji hypokotylu, koreňa a v procesoch kvitnutia. Zároveň boli zhotovené in silico analýzy za účelom predikcie štruktúry, transkripčnej regulácie a homológie jednotlivých proteínov a génov. Prítomnosť úspešne pripraveného konštruktu AtUBQ10::YFP:cMMK3 bola v rastlinách A. thaliana (Col 0) potvrdená metódou PCR genotypizácie a metódou Western-blot v rastline M. sativa (RSY).

Abstract:

The term mitogen activated protein kinases includes signaling proteins responsible for transmission of extracellular signals and regulation of cellular processes. Their importance results from the sessile nature of plant organisms, which are exposed to adverse environmental conditions and MAP kinase cascade represents one of the ways of defense. In this bachelor thesis, constructs of MMK3 protein in fusion with fluorescent protein were prepared. The constructs were validated using method of transient transformation of Nicotiana benthamiana leaves and the Western-blot method. Subsequently were used for stable transformation in Medicago sativa (RSY) and also heterologically in Arabidopsis thaliana (Col-0, mpk13). Transgenic plants were verified by PCR genotyping or Western blot analysis. Furthermore, phenotypic analyses of A. thaliana mpk13 mutant lines were performed, which pointed to differences in hypocotyl, root development and flowering processes. In silico analyses were also performed in order to predict the structure, transcriptional regulation and homology of individual proteins and genes. Presence of succesfully prepared construct AtUBQ10::YFP:cMMK3, was detected in A. thaliana plants (Col 0) by PCR genotyping and by Western-blot in M. sativa (RSY) plant.

Keywords

Lucerna siata, MMK3, MPK13, arábkovka Thalova, in silico analýzy

Language used: Czech

Date on which the thesis was submitted / produced: 6. 5. 2024

Thesis defence

• Supervisor: Mgr. Ivan Luptovčiak, Ph.D.

Palacký University Olomouc

Faculty of Science

Bachelor programme / field:
Biotechnology and Genetic Engineering / Biotechnology and Genetic Engineering