Optimizacija izolacije zigotnih embrija uročnjaka (Arabidopsis thaliana (L.) Heynh.) u svrhu istraživanja kvantitativne ekspresije gena

master's thesis

• Title (english): Optimization of Arabidopsis thaliana (L.) Heynh. zygotic embryo isolation for subsequent use in quantitative gene expression analysis
• Author: Tanita Demir
• Mentor: Nataša Bauer (mentor)
• Committee member: Nataša Bauer (predsjednik povjerenstva)
• Committee member: Martina Šeruga Musić (član povjerenstva)
• Committee member: Željka Vidaković-Cifrek (član povjerenstva)
• Committee member: Dunja Leljak-Levanić (član povjerenstva)
• Granter: University of Zagreb; Faculty of Science; (Department of Biology); Zagreb
• Defense date and country: 2021-02-26, Croatia
• Scientific / art field, discipline and subdiscipline: NATURAL SCIENCES; Biology

Abstract

Analize genske ekspresije u pojedinim embrionalnim stadijima daju uvid u genetske mehanizme i regulatorne mreže koji omogućuju potpuni razvoj embrija. Zbog točno određenog i predvidljivog razvoja tijekom embriogeneze, uročnjak (Arabidopsis thaliana (L.) Heynh.) je idealan model u takvim istraživanjima. Brojne tehničke poteškoće povezane s izolacijom različitih stadija zigotnih embrija otežavaju analize ekspresije gena tijekom embriogeneze. Ovim radom uhodane su lako primjenjive metode izolacije pojedinih stadija embrija uročnjaka koje omogućuju izdvajanje velikog broja čistih embrija, oslobođenih od stanica majčinske biljke koje u ovakvom tipu istraživanja predstavljaju kontaminaciju. Uhodan je postupak mikromanipulacije zigotnih embrija, upotrebom invertnog mikroskopa s mikrodisektorom, koji omogućuje izdvajanje nekoliko razvojnih stadija embrija. Na različitim transgeničnim i mutantnim linijama uročnjaka provedena je metoda izolacije dovoljne količine mRNA za analize ekspresije gena u kotiledonarnim embrijima. Metodom reverzne transkripcije mRNA su prepisane u cDNA koja je korištena kao kalup za umnažanje u kvantitativnoj lančanoj reakciji polimeraze (qPCR), te je analizirana relativna ekspresija gena CML41 i FBW2 u kotiledonarnim embrijima. Provedeno istraživanje ukazuje da je za analizu genske ekspresije do 6 gena metodom qPCR potrebno izolirati ukupnu mRNA iz 70-tak kotiledonarnih embrija. Metode izolacije embrija i mRNA provedene u ovom radu pomoći će u planiranju eksperimenata transkriptomske analize embrija u budućim istraživanjima.

Abstract (english)

Gene expression analyses at individual embryonic stages give insight into genetic mechanisms and regulatory networks which enable completion of embryonic development. Due to its precise and predictable pattern of embryogenesis, Arabidopsis thaliana (L.) Heynh. represents an ideal model plant. Numerous technical difficulties of zygotic embryo isolation at different stages hinder gene expression analyses during embryogenesis. Here, easily applicable methods of A. thaliana embryo isolation were described, enabling separation of a large number of embryos clean from surrounding tissues. A micromanipulation procedure using inverted microscope with microdissector was optimized, enabling isolation of embryos at different developmental stages. Furthermore, a method of mRNA isolation from cotyledonary embryos of different transgenic and mutant lines in quantities sufficient for subsequent gene expression analysis was performed. Using reverse transcription method, equivalent quantities of mRNA were transcribed to cDNA, which was used as template for amplification in quantitative polymerase chain reaction (qPCR) and relative gene expression levels of CML41 and FBW2 in cotyledonary embryos were analysed. This research shows that total mRNA isolated from 70 cotyledonary embryos is sufficient for quantitative gene expression analysis by qPCR of at least 6 genes. Embrio and mRNA isolation methods performed in this research will help in future planning of embryo transcriptome analyses.

• Keywords: Arabidopsis thaliana; izolacija embrija; mikromanipulacija; izolacija mRNA; qPCR
• Keywords (english): Arabidopsis thaliana; embryo isolation; micromanipulation; mRNA isolation; qPCR
• Language: croatian
• URN:NBN: urn:nbn:hr:217:483614
• 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: 2021-03-09 08:08:59