magistrsko delo
Nina Kobe (Author), Kristina Gruden (Mentor), Brigita Lenarčič (Thesis defence commission member), Marina Klemenčič (Thesis defence commission member), Marko Dolinar (Co-mentor)

Abstract

Biotski stres v rastlinah sproži obrambni odgovor. V uravnavanje slednjega so vključene signalizacijske poti s fitohormoni, med katerimi so najpomembnejši salicilna kislina (SA), jasmonska kislina (JA) in etilen (ET). Spremljanje prostorsko-časovne dinamike aktivacije genov določene signalne poti in s tem proučevanje rastlinskega odziva na stres nam omogočajo genetsko kodirani biosenzorji. V prvem delu magistrskega dela smo med štirimi nativnimi promotorji genov TGA2_R, TGA2_PW, PR1b_R in PR1b_D iz krompirja izbrali najprimernejšega za uporabo v biosenzorju signalne poti SA. Za ta namen smo promotorska zaporedja sklopili s kodirajočim zaporedjem za luciferazo. Aktivnost promotorjev ob tretiranju s SA, JA in ET smo po prehodni transformaciji tobaka Nicotiana benthamiana preverjali z luciferaznim testom in na podlagi rezultatov izbrali promotor TGA2_R, ki se je izkazal kot najbolj selektiven za SA in s tem najprimernejši za uporabo v biosenzorju. V drugem delu smo optimizirali začetni korak pri metodi za določanje odzivnosti promotorjev v krompirju, to je pripravo protoplastov. Izolirali smo protoplaste krompirja kultivarjev Rywal in Désirée po treh različnih protokolih, izbrali najprimernejšega za nadaljnjo transfekcijo protoplastov in ga optimizirali. Ugotovili smo, da je za izolacijo protoplastov optimalna starost rastlin krompirja pri kultivarju Rywal 3–4 tedne. V naslednjem koraku bomo izbrani promotor uporabili za stabilno transformacijo rastlin krompirja in bomo s tem bližje razumevanju odziva rastlin krompirja na stresorje, kar bo pripomoglo k pripravi odpornejših sort.

Keywords

krompir;bolezni krompirja;škodljivci krompirja;rastlinski imunski odziv;biosenzorji rastlinskega imunskega odziva;gensko kodiran biosenzor;protein luciferaza;luciferazni test aktivnosti promotorjev;salicilna kislina;izolacija protoplastov krompirja;magistrska dela;

Data

Language: Slovenian
Year of publishing:
Typology: 2.09 - Master's Thesis
Organization: UL FKKT - Faculty of Chemistry and Chemical Technology
Publisher: [N. Kobe]
UDC: 577.27:581.1(043.2)
COBISS: 81994755 Link will open in a new window
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Downloads: 72
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Other data

Secondary language: English
Secondary title: Optimisation of methodology for studying gene promoter activity in potato
Secondary abstract: Biotic stress triggers an immune response in plants. The regulation of the immune response involves signaling pathways involving phytohormones, of which salicylic acid (SA), jasmonic acid (JA), and ethylene (ET) are the most important. Monitoring of the spatiotemporal dynamics of the gene activation under a particular signaling pathway, and thus studying plant's stress response is made possible by using genetically encoded biosensors. In the first part of the Master's thesis, we selected the most suitable biosensor of the SA signaling pathway in potato among four native promoters of genes TGA2_R, TGA2_PW, PR1b_R and PR1b_D. For this purpose, the promoter sequences were fused with luciferase coding sequence. Promoter activity upon treatment with SA, JA and ET was tested after transient transformation of tobacco plants (Nicotiana benthamiana) by luciferase assay. Thus, we selected TGA2_R as the most SA-specific promoter for use in a biosensor. In the second part of the thesis we optimized the first step of the method for determining promoter responsiveness in potato that is isolation of protoplasts. We isolated protoplasts from Rywal and Désirée potato cultivars according to three different protocols, and selected and optimized the most suitable one for protoplast transfection. We showed that 3–4 weeks old Rywal plants are optimal for isolation of potato protoplasts. In the future, the selected promoter will be used for stable transformation of potato. Its use will lead us closer to understanding of potato plant response to stressors, which will aid in development of more robust cultivars.
Secondary keywords: plant immune response;genetically encoded biosensor;luciferase test of promoter activity;salicilic acid;isolation of potato protoplasts;
Type (COBISS): Master's thesis/paper
Study programme: 1000377
Embargo end date (OpenAIRE): 1970-01-01
Thesis comment: Univ. v Ljubljani, Fak. za kemijo in kemijsko tehnologijo, smer Biokemija
Pages: 147 str.
ID: 13342282