Strategija razvoja biosenzorjev imunskega odziva v krompirju

Levak, Valentina (author), Gruden, Kristina (mentor), Dolinar, Marko (comentor)

Kadar rastlina doživi stres, naj bo biotskega ali abiotskega izvora, v ogroženih celicah pride do imunskega odziva. Informacija o vrsti stresa se prenese med celicami s hormoni in drugimi signalnimi molekulami, ki nastajajo med celičnim imunskim odzivom. Imunski odziv lahko spremljamo v tkivu skozi čas preko aktivnosti promotorjev genov, ki so vključeni v regulatorno omrežje imunskega odgovora. Z izbiro promotorja gena iz določene signalne poti imunskega odziva lahko sledimo prostorsko-časovni dinamiki aktivacije izbranih genov in s tem širjenju imunskega odgovora. V magistrskem delu smo preverjali ustreznost treh metod za testiranje nativnih inducibilnih rastlinskih promotorjev po prehodni transformaciji tobaka Nicotiana benthamiana. Te metode so: kvalitativni test z glukuronidazo, kvantitativni test z luciferazo in konfokalna makroskopija. Ugotovili smo, da je test z luciferazo najprimernejši za ta namen, saj je hiter, občutljiv in ponovljiv ter omogoča spremljanje aktivnosti promotorja in vivo pri večjem številu vzorcev in skozi čas. Test smo nato optimizirali za naš namen. Reporterske proteine, ki smo jih uporabili za preverjanje metod, smo izražali pod kontrolo nativnih promotorjev genov PR1b, Pti5, MC in CPI8. Prvi trije so bili iz genomske DNA krompirja (Solanum tuberosum) že pomnoženi, promotorska zaporedja CPI8 pa smo pomnožili iz genoma kultivarjev krompirja Rywal in Désirée na podlagi referenčnega zaporedja podvrste Phureja ter analizirali predvidene cis-regulatorne regije. Aktivnost izbranih različic promotorjev CPI8 ob indukciji z jasmonsko kislino smo preverjali po prehodni transformaciji tobaka z optimiziranim luciferaznim testom. Različico, pri kateri je bilo izražanje luciferaze najmočnejše, smo izbrali za biosenzor signalne poti jasmonske kisline in ga bomo uporabili za stabilno transformacijo rastlin krompirja za nadaljnje proučevanje rastlinskega odziva na stres. Nova spoznanja o nastanku in razširjanju imunskega odziva pri rastlinah nam pomagajo razumeti potek rastlinskih bolezni in rastlinskega odgovora na kombinacije različnih stresorjev. To razumevanje je ključno pri pripravi novih, odpornejših sort.

• Language: Slovenian
• Keywords: rastlinski imunski odziv, proteinazni inhibitor, luciferazni test aktivnosti promotorjev, jasmonska kislina, transkripcijska regulacija
• Work type: Master's thesis/paper
• Typology: 2.09 - Master's Thesis
• Organization: FKKT - Faculty of Chemistry and Chemical Technology
• Year: 2020
• PID: 20.500.12556/RUL-119691
• COBISS.SI-ID: 31298051
• Publication date in RUL: 10.09.2020

Secondary language

• Language: English
• Title: Strategy for development of immune response biosensors in potato

Abstract

In biotic or abiotic stress, immune response is activated in affected plant cells. Information about the type of stress is shared between cells through hormones and other signal molecules synthesized in affected cells. Immune response can be followed through the activity of promoters of the genes that are involved in the immune response regulatory network. With the selection of a gene promoter from a specific immune response signaling pathway, we can track spatiotemporal dynamics of the activation of chosen genes and therewith, the expansion of the immune response. In this Master thesis, the applicability of three different methods for testing native inducible plant promoters after transient transformation of tobacco plants Nicotiana benthamiana was tested. These methods are: qualitative beta-glucuronidase assay, quantitative luciferase assay and confocal macroscopy. The luciferase assay was shown to be the most suitable for our purposes as it is fast, sensitive and repeatable and allows measuring of promoter activity in vivo in higher number of samples and through time. The assay was optimized in order to serve our purposes. Reporter proteins that were used in methods testing were expressed under control of native promoters of genes PR1b, Pti5, MC and CPI8. The first three had already been amplified from potato (Solanum tuberosum) genomic DNA, while promoter regions of CPI8 were amplified from the genome of cultivars Rywal and Désirée according to the reference sequence in Phureja subspecies. Afterwards, predicted cis-regulatory regions were analyzed in the amplified promoter regions. Actual activities of three chosen variants of CPI8 promoters were tested for inducibility to jasmonic acid after transient transformation of tobacco in the optimized luciferase assay. A variant that showed the highest luciferase expression was chosen for a biosensor of jasmonic acid signaling pathway and will be used for stable transformation of potato plants for further studies on plant stress response. New discoveries about the start and expansion of immune response in plants help us understand the course of plants’ diseases and response to the combination of different stressors. This understanding is key in the preparation of new, more robust plant cultivars.

• Keywords: plant immune response, proteinase inhibitor, luciferase test of promoter activity, jasmonic acid, transcriptional regulation