Štúdium proteín-proteínových interakcií a príprava multiplex CRISPR/Cas9 vektorov za účelom mutagenézy génov u lucerny siatej (Medicago sativa L.)

Bc. Peter ŠÁLY

Master's thesis

Protein-protein interaction studies and preparation of multiplex CRISPR/Cas9 vectors for alfalfa (Medicago sativa L.) mutagenesis

Abstract:

Symbióza s pôdnymi baktériami fixujúcimi dusík je významnou poľnohospodárskou vlastnosťou pri pestovaní lucerny siatej. Nielenže nepotrebuje byť hnojená, ale navyše dochádza k obohacovaniu pôdy o dusík. To vedie k záujmu o štúdium a pochopenie molekulárneho procesu zabezpečujúceho symbiózu. MAPK a ROP GTPázy sa javia byť súčasťou signálnej dráhy potrebnej na vytvorenie symbiotickej interakcie. V tejto diplomovej práci boli teoreticky popísané multiplex CRISPR/Cas9 systémy a ukázaný chronologický progres aplikácie CRISPR/Cas9 metódy na lucerne siatej za posledné roky. Taktiež bola charakterizovaná funkcia a štruktúra ROP GTPáz a ich možného prepojenia s MAPK. V neposlednom rade boli predstavené metódy pre štúdium proteín-proteínových interakcií, a to konkrétne bimolekulárna fluorescenčná komplementácia a kvasinkový dvojhybridný systém. V experimentálnej časti boli pripravené multiplex CRISPR/Cas9 konštrukty pre editáciu SIMK a SIMKK génov, ktoré boli stabilne transformované do lucerny siatej. Mikroskopicky vyselektované transgénne rastliny boli podrobené Western blot analýze a analýze polymerázou reťazovou reakciou s restrikčným štiepením. Následne došlo k charakterizácii povahy typu mutácií transgénnej rastliny a bola získaná lucerna siata editovaná v géne SIMK. V druhej časti práce boli pripravené vektory pre bimolekulárnu fluorescenčnú komplementáciu a kvasinkový dvojhybridný systém a ich realizáciou bola zistená interakcia medzi proteínmi ROP8 a SIMK. Bolo zistené možné prepojenie ROP GTPázovej a MAPK signálnej dráhy.

Abstract:

Symbiosis with nitrogen-fixing soil bacteria is an important agricultural feature in the cultivation of alfalfa. Not only does it not need to be fertilized, but it also enriches the soil with nitrogen. This leads to an interest in studying and understanding the molecular process behind symbiosis. MAPK and ROP GTPases appear to be part of the signaling pathway required to establish the symbiotic interaction. In this diploma thesis, multiplex CRISPR/Cas9 systems were theoretically described and the chronological progress of the CRISPR/Cas9 method application on alfalfa over the last years was shown. The function and structure of ROP GTPases and their possible connection with MAPK were also characterized. Last but not least, methods for the study of protein-protein interactions were presented, namely bimolecular fluorescent complementation and the yeast two-hybrid system. In the experimental part, multiplex CRISPR/Cas9 constructs were prepared for the editing of SIMK and SIMKK genes, which were stably transformed into alfalfa. Microscopically selected transgenic plants were subjected to Western blot analysis and analysis by polymerase chain reaction with restriction digestion. Subsequently, the nature of the mutation type of the transgenic plant was characterized and was obtained alfalfa edited in the SIMK gene. In the second part of the thesis, vectors for bimolecular fluorescent complementation and the yeast two-hybrid system were prepared, and by their implementation, the interaction between the ROP8 and SIMK proteins was detected. A possible link between the ROP GTPase and MAPK signaling pathways has been identified.

Keywords

BiFC, lucerna siata, multiplex CRISPR/Cas9, ROP8, ROP GTPázy, SIMK, SIMKK, Y2H

Language used: Czech

Date on which the thesis was submitted / produced: 29. 4. 2024

Thesis defence

• Supervisor: Mgr. Miroslava Medelská, Ph.D.

Palacký University Olomouc

Faculty of Science

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