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URAVNAVANJE PREPISOVANJA IN LOKALIZACIJE TRANSKRIPCIJSKEGA FAKTORJA PTI
Fric, Katja (Author), Gruden, Kristina (Mentor) More about this mentor... This link opens in a new window, Dermastia, Marina (Reviewer)

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Abstract
Po okužbi krompirja s krompirjevim virusom PVY se transkripcijski faktor Pti5 premakne v jedro. Pti5 iz družine ERF (angl. ethylene response factors) ima glavno vlogo pri aktivaciji in utišanju tarčnih genov, ki so pomembni v odgovoru na stres. V magistrskem delu smo raziskovali mehanizem, ki sproži lokalizacijo Pti5 v jedro. Modelne rastline tobaka Nicotiana benthamiana smo prehodno transformirali s konstruktom, ki vsebuje gen Pti5 v fuziji z rumenim fluorescenčnim proteinom (YFP) pod konstitutivnim promotorjem 35S in jih obdelali s kinaznim inhibitorjem. Sledili smo izražanju fluorescenčnega proteina s pomočjo konfokalnega mikroskopa. Iz dobljenih rezultatov sklepamo, da sama translokacija Pti5 ni zaradi fosforilacije. Prav tako nas je zanimalo, če je za translokacijo dovolj le en virusni protein. Ugotovili smo, da lahko že P19, virusni supresor utišanja RNA iz tombusvirusov vpliva na translokacijo Pti5 v jedro. Vedeli smo, da je Pti5 na stičišču hormonskih poti salicilne kisline (SA), etilena (ET) in jasmonske kisline (JA). Z uporabo β-glukuronidaznega testa smo ugotavljali aktivnost promotorja Pti5 v primerjavi s promotorjem PR1b, ki je označevalec signalne poti SA, po obdelavi z zgoraj naštetimi hormoni oz. njihovimi inhibitorji. Sklepamo, da tako analog SA INA, kot prekurzor ET ACC povečata indukcijo promotorja Pti5 in PR1b v listih tobala N. benthamiana. Prav tako iz rezultatov sklepamo, da PR1b uravnava SA neodvisno od ET. Pti5 najverjetneje uravnava SA v etilenski signalni poti. S prenosom western smo želeli ugotoviti, če se pri rastlinah okuženih s PVY, poveča kopičenje Pti5 v jedru. Slednje smo potrdili le z rezultati s konfokalnim mikroskopom.

Language:Slovenian
Keywords:Transkripcijski faktor/Pti5/ PR1b/krompir (Solanum tuberosum L.)/Nicotiana benthamiana/&#946, - glukuronidazni test/konfokalni mikroskop
Work type:Master's thesis/paper (mb22)
Organization:BF - Biotechnical Faculty
Year:2017
COBISS.SI-ID:4477263 This link opens in a new window
Views:1202
Downloads:463
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Secondary language

Language:English
Title:REGULATION OF TRANSCRIPTION AND LOCALIZATION OF TRANSCRIPTION FACTOR PTI
Abstract:
Following the infection of the potato, with the potato virus Y (PVY), transcription factor Pti5 translocates into nucleus. Pti5 belongs to ERF familiy (ethylene response factor), which have the main role in activation and silencing of targeted genes important in plant response to stress. In this master thesis, we were studying which mechanism is responsible for translocation of Pti5 into the nucleus. Using transient transformation of Nicotiana benthamiana, with the construct, which has gene Pti5 fused with yellow fluorescent protein (YFP) under constitutive 35S promoter and then treated the plants with kinase inhibitor. We followed the expression of YFP under confocal microscope. Our results show that translocation of Pti5 is not caused by phosphorylation. Additionally, we found, that only one protein P19, viral supressor of RNA silencing from tombusviruses, is enough for translocation of Pti5 to the nucleus. We knew that Pti5 is in the crossroad between signaling pathways of salycilic acid (SA), ethylen (ET) and jasmonic acid (JA). We were interested in activity of promoter Pti5, in comparison to promoter PR1b, which is marker of SA signaling pathway, following treatment with hormones and their inhibitors mentioned above. Our results show that both, analog of SA INA and precursor of ET ACC, increase the induction of promoter Pti5 and PR1b in N. benthamiana leaves. We also can conclud that PR1b is regulated by SA independantly of ET, while induction of Pti5 by SA is most likely regulated through ET signaling. With Western blot we wanted to found out, if the accumulation of Pti5 is increased in the plants infected with PVY. We proved that only with the results obtained by confocal microscopy.

Keywords:Transcripton factor/Pti5/PR1b/krompir (Solanum tuberosum L.) /Nicotiana benthamiana/transient transformation/GUS assay/confocal microscope

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