Selection of Arabidopsis thaliana plant lines for the study of single locus proteomics
Author
Bolsi, Nicholas
Term
4. term
Education
Publication year
2023
Submitted on
2023-06-01
Pages
43
Abstract
This thesis establishes Arabidopsis thaliana lines and validation assays to enable single‑locus proteomics of defined transcriptional contexts. Focusing on RNA polymerase II transcription and the role of histone post‑translational modifications, it motivates the mapping of protein assemblies at promoters, elongation regions, and a model of tandem transcriptional interference (tTI) caused by a T‑DNA insertion at the QUA1 locus. The qua1‑1 allele provides clear molecular and phenotypic readouts—including dwarfism, defective cell adhesion, and Ruthenium red staining—to anchor locus‑specific studies of proteins associated with interfering transcription. The work reviews strategies for targeting discrete genomic sites (zinc fingers, TALEs, dCas9) and outlines a practical pipeline from plant growth to proteome analysis: homozygous line selection, PCR‑based genotyping, protein detection by Western blot, phenotypic assays such as hypocotyl length and Ruthenium red staining, and the selection of appropriate negative controls to manage signal‑to‑noise in affinity purifications. By prioritizing rigorous line selection and controls, the project lays the groundwork for mass‑spectrometry‑based identification of proteins at single genomic loci, both at tTI regions and at transcription initiation and elongation sites. Specific proteomic findings are not presented in the introductory material provided, but the thesis defines the experimental framework required for their acquisition.
Dette speciale etablerer Arabidopsis thaliana‑linjer og valideringsassays til at muliggøre single‑lokus proteomik af veldefinerede transkriptionskontekster. Med fokus på RNA‑polymerase II‑transkription og betydningen af histon‑posttranslationelle modifikationer motiveres kortlægning af proteinkomplekser ved promotorer, elongationsområder og en model for tandem transkriptionsinterferens (tTI) forårsaget af en T‑DNA‑indsættelse ved QUA1‑locus. Allelen qua1‑1 giver tydelige molekylære og fænotypiske readouts—herunder dværgvækst, defekt celladhæsion og Ruthenium red‑farvning—som kan forankre lokus‑specifikke studier af proteiner knyttet til interfererende transkription. Arbejdet gennemgår strategier til målretning af diskrete genomiske steder (zinkfingre, TALEs, dCas9) og skitserer en praktisk pipeline fra plantevækst til proteomeanalyse: udvælgelse af homozygote linjer, PCR‑baseret genotypning, proteindetektion med Western blot, fænotypiske assays som hypokotyllængde og Ruthenium red‑farvning samt udvælgelse af passende negative kontroller for at håndtere signal‑støj i affinitetsoprensninger. Ved at prioritere stringent linjeudvælgelse og kontroller lægger projektet grunden for massespektrometrisk identifikation af proteiner ved enkelte genomiske loci, både i tTI‑regioner og ved transkriptions‑initierings‑ og elongationssteder. Konkrete proteomiske fund præsenteres ikke i det tilgængelige introduktionsmateriale, men specialet definerer den eksperimentelle ramme, der kræves for at opnå dem.
[This apstract has been generated with the help of AI directly from the project full text]
Keywords