Publicação
Use of the MS2 system for in vivo mRNA visualization in plant cells
| dc.contributor.author | Novita, Marina Melucci | |
| dc.contributor.institution | Faculty of Sciences | |
| dc.contributor.supervisor | Teixeira, Rita Teresa Pereira | |
| dc.date.accessioned | 2026-02-19T17:25:01Z | |
| dc.date.available | 2026-02-19T17:25:01Z | |
| dc.date.issued | 2026 | |
| dc.description | Tese de mestrado, Biologia Molecular e Genética, 2026, Universidade de Lisboa, Faculdade de Ciências | |
| dc.description.abstract | To coordinate developmental processes and transmit environmental stimuli, plants recruit a variety of molecules, including mobile mRNAs, as systemic signals for inter-organ communication. Despite the growing number of transcripts classified as mobile, the mechanisms and the extent of their movement remain poorly understood. Given the involvement of the GI and PIP5K2 genes in processes that involve communication between tissues, we hypothesized that their mRNAs exhibit intercellular or long-distance transport. With the goal of investigating mRNA trafficking in plants, we began implementing the MS2 tagging system for real-time visualization of GI and PIP5K2 mRNAs. In this technique, the target RNA is tagged with stem–loop (SL) repeats, a structure responsible for recruiting the MS2 protein. By co-expressing MS2 fused to a fluorescent reporter and the SL-tagged mRNA, the transcript’s localization can be observed under a confocal microscope. Here we describe a suggested pipeline for cloning GI and PIP5K2 into the MS2 system. However, due to time constraints, an alternative plan was adopted, replacing these two genes with FT for in vivo visualization of mRNA in Nicotiana benthamiana leaves through Agrobacterium tumefaciens–mediated transient expression. In the initial cloning steps, we identified limitations such as the impact of insert size on cDNA isolation and amplification efficiency. In addition, specific PCR amplification protocols were established for GI and PIP5K2 genes. The results obtained confirm the feasibility of the MS2 system for tracking FT mRNAs in N. benthamiana; however, the overexpression artifacts observed required methodological adjustments to ensure detectable and consistent expression levels. In the fluorescence images, we demonstrate that FT mRNA moves intracellularly and accumulates mainly at plasmodesmata sites. Finally, by overcoming cytoplasmic fluorescent noise—the main barrier to the use of the MS2 system in plants—the method becomes promising for tracking mRNAs with restricted and dynamic expression. | en |
| dc.format | application/pdf | |
| dc.identifier.uri | http://hdl.handle.net/10400.5/117214 | |
| dc.language.iso | eng | |
| dc.subject | MS2 | |
| dc.subject | Mobile mRNA | |
| dc.subject | in vivo imaging | |
| dc.subject | Confocal microscopy | |
| dc.subject | Plant biology | |
| dc.title | Use of the MS2 system for in vivo mRNA visualization in plant cells | en |
| dc.type | master thesis | |
| dspace.entity.type | Publication | |
| rcaap.rights | openAccess |
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