Retrotranspozon Tto1 kao alat za ciljanu inaktivaciju gena uročnjaka (Arabidopsis thaliana)

master's thesis

• Title (english): Retrotransposon Tto1 as a tool for gene targeting in Arabidopsis thaliana
• Author: Branka Popović
• Mentor: Višnja Besendorfer (mentor)
• Mentor: Nenad Malenica (pomoć pri izradi, neposredni voditelj)
• Committee member: Sandra Radić Brkanac (predsjednik povjerenstva)
• Committee member: Damjan Franjević (član povjerenstva)
• Granter: University of Zagreb; Faculty of Science; (Department of Biology); Zagreb
• Defense date and country: 2010, Croatia
• Scientific / art field, discipline and subdiscipline: NATURAL SCIENCES; Biology

Abstract

Ciljana inaktivacija gena počela se primjenjivati na miševima 80-ih godina prošlog stoljeća. Uspješnost ove strategije temelji se na relativno visokoj učestalosti homologne rekombinacije u somatskim stanicama miša i učinkovitom sustavu pozitivno-negativne selekcije. S druge strane, u biljkama se ugradnja strane DNA odvija pretežno procesom ilegitimne rekombinacije. Ovim radom započeto je istraživanje mogućnosti ciljane inaktivacije gena biljke-modela Arabidopsis thaliana (uročnjak) upotrebom retrotranspozona duhana Tto1. Ideja se temelji na manipulaciji procesa reverzne transkripcije, ključnog koraka u životnom ciklusu retrotranspozona, u svrhu proizvodnje supstrata (cDNA) za homolognu rekombinaciju. Transfomacijom bakterijama vrste Agrobacterium tumefaciens stvoreno je nekoliko transgenih linija uročnjaka s različitim varijantama retrotranspozona Tto1. Pomoću tih linija željelo se istražiti da li se Tto1 prepisuje u novom domaćinu te da li može završiti životni ciklus s ugrađenim genom biljegom. U slučaju pozitivnog ishoda, pristupilo bi se zamjeni gena biljega za gen uročnjaka koji želimo ciljano inaktivirati. Pokazano je da su sve priređene varijante Tto1 transkripcijski aktivne u uročnjaku, te da se nestabilnost Tto1 binarnih vektora u agrobakterijama, opisana u literaturi, ne ispoljava u uvjetima kratkotrajnog kultiviranja. Također, preliminarni rezultati na temelju fenotipske ekspresije gena biljega pokazali su da Tto1 može završiti životni ciklus. Međutim, dosadašnjim istraživanjima nije se uspjelo dokazati nove kopije Tto1 u genomu uročnjaka.

Abstract (english)

Gene targeting was first applied on mice during the 80-ies of the last century. The success of this strategy is based on a relatively high frequency of homologous recombination in mouse somatic cells and an effective system of positive-negative selection. On the other hand, in plants the integration of exogenous DNA occurs predominantly by illegitimate recombination. In this work we studied the possibility of gene targeting in Arabidopsis thaliana using tobacco retrotransposon Tto1. The idea is based on the manipulation of the process of reverse transcription, a key step in the life cycle of retrotransposon, for production of substrate (cDNA) for homologous recombination. By applying Agrobacterium tumefaciens-mediated transformation several transgenic lines of A. thaliana with different variants of retrotransposon Tto1 were created. Using these lines it was explored whether Tto1 was transcripted into the new host, and whether Tto1 can complete the life cycle with an integrated marker gene. In the case of positive outcome, we will approach to replacing gene marker for a gene that we want to specifically inactivate. It was shown that all variants of cloned Tto1 were transcriptionally active and previously described instability of Tto1 binary vectors in Agrobacterium tumefaciens didn't manifest itself in short-term cultivation. Preliminary results based on gene marker expression, have shown that a Tto1 can complete its life cycle. However, so far new copies were not detected in the genome of transformed A.thaliana plants.

• Keywords: Arabidopsis thaliana; uročnjak; ciljana inaktivacija gena; Tto1
• Keywords (english): Arabidopsis thaliana; gene targeting; Tto1
• Language: croatian
• URN:NBN: urn:nbn:hr:217:269084
• Study programme: Title: Biology; specializations in: Molecular biology; Course: Molecular biology; Study programme type: university; Study level: graduate; Academic / professional title: magistar/magistra biologije (magistar/magistra biologije)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access
• Created on: 2018-03-29 09:05:12