Istraživanje interakcija proteina BPM1, DMS3 i RDM1 bimolekulskom fluorescencijskom komplementacijom u stanicama duhana

master's thesis

• Title (english): Protein interaction analysis of BPM1, DMS3 and RDM1 in tobacco cells using bimolecular fluorescent complementation
• Author: Toni Rendulić
• Mentor: Nataša Bauer (mentor)
• Committee member: Nataša Bauer (član povjerenstva)
• Committee member: Željka Vidaković-Cifrek (član povjerenstva)
• Committee member: Martina Šeruga Musić (član povjerenstva)
• Committee member: Dunja Leljak-Levanić (član povjerenstva)
• Granter: University of Zagreb; Faculty of Science; (Department of Biology); Zagreb
• Defense date and country: 2018-04-17, Croatia
• Scientific / art field, discipline and subdiscipline: NATURAL SCIENCES; Biology

Abstract

Proteini BPM sudjeluju u regulaciji nekoliko transkripcijskih faktora kroz ubikvitinsko-proteasomski put. Proteini DMS3 i RDM1 su važne komponente sustava za RNA-posredovanu metilaciju DNA (RdDM). U preliminarnom istraživanju pokazalo se da su DMS3 i RDM1 potencijalni interakcijski partneri proteina BPM1. U ovom radu su istražene interakcije proteina BPM1, DMS3 i RDM1 pomoću bimolekulske fluorescencijske komplementacije (BiFC). Radi provedbe analize BiFC, generirani su prikladni plazmidni konstrukti tehnikom kloniranja In-Fusion. Stanice epiderme duhana Nicotiana benthamiana, epiderme luka Allium cepa i stanice duhana BY-2 transformirane su generiranim plazmidnim konstruktima pomoću agroinfiltracije ili bombardiranjem mikročesticama zlata. Detekcija proteinskih interakcija provedena je fluorescencijskom mikroskopijom. DMS3 i RDM1 su imali interakciju u svim istraživanim stanicama, dok je formacija dimera DMS3 detektirana u epidermalnim stanicama duhana N. benthamiana i luka A. cepa. Interakcija proteina BPM1 i DMS3 je potvrđena u duhanu N. benthamiana i u stanicama BY-2. Interakcija između BPM1 i RDM1 nije detektirana u ovom radu. Potrebna su dodatna istraživanja kako bi se bolje razumjele pojedinosti interakcije između BPM1 i DMS3 te potencijalni utjecaj BPM1 na RdDM.

Abstract (english)

BPM proteins are known to participate in the regulation of several transcription factors via the ubiquitin proteasome pathway. DMS3 and RDM1 proteins are important components of the RNA-directed DNA methylation (RdDM) machinery. In preliminary research, DMS3 and RDM1 were shown to be potential interactors of BPM1. Here, interactions of BPM1, DMS3 and RDM1 proteins were analyzed via bimolecular fluorescence complementation (BiFC). To perform BiFC analysis, suitable plasmid constructs were generated using In-Fusion cloning technique. Nicotiana benthamiana, Allium cepa and BY-2 cells were transiently transformed with the generated constructs via agroinfiltration or microparticle bombardment. Finally, fluorescence microscopy was used to detect and analyze protein interactions. DMS3 and RDM1 interacted in all examined cells, while DMS3 dimer formation was detected in N. benthamiana and A. cepa epidermal cells. Interaction between BPM1 and DMS3 was proved in N. benthamiana leaf epiderm and in tobacco BY-2 cells. By using BiFC, no interaction was detected between BPM1 and RDM1 in this study. Further research is required to better understand the details of interaction between BPM1 and DMS3 proteins and the potential effect of BPM1 on RdDM.

• Keywords: BTB; MATH; RdDM; BiFC; transformacija
• Keywords (english): BTB; MATH; RdDM; BiFC; transformation
• Language: croatian
• URN:NBN: urn:nbn:hr:217:006801
• Study programme: Title: Molecular Biology; Study programme type: university; Study level: graduate; Academic / professional title: magistar/magistra molekularne biologije (magistar/magistra molekularne biologije)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access
• Created on: 2018-05-03 08:34:57