Auksin-hidrolaze: analiza i regeneracija mutanata uročnjaka (Arabidopsis thaliana L.)

master's thesis

• Title (english): Auxin-hydrolases: analysis and regeneration of Arabidopsis thaliana L. mutants
• Author: Amel Zulji
• Mentor: Nataša Bauer (mentor)
• Committee member: Nataša Bauer (predsjednik povjerenstva)
• Committee member: Marin Ježić (član povjerenstva)
• Committee member: Ivana Šola (član povjerenstva)
• Granter: University of Zagreb; Faculty of Science; (Department of Biology); Zagreb
• Defense date and country: 2017-02-27, Croatia
• Scientific / art field, discipline and subdiscipline: NATURAL SCIENCES; Biology

Abstract

U ovom radu su opisani mehanizmi biosinteze, transporta i konjugacije indol-3-octene kiseline (IAA), kao dominantnog oblika auksina, i njen učinak na razvojne i fiziološke procese, s posebnim osvrtom na spoznaje o detaljima tih procesa kod eksperimentalne biljke Arabidopsis thaliana. Tehnologijom in-fusion sintetiziran je binarni vektor pHOV:AtILL2-GFP, te su metodom floral dip regenerirane transgenične biljke A. thaliana ekotip WS i Col-0 s pojačanom ekspresijom gena AtILL2 i rekombinantnog gena AtILL2-GFP. Lokalizacija fuzijskog proteina AtILL2-GFP, praćena fluorescencijskom mikroskopijom, sugerira endoplazmatski retikulum kao mjesto djelovanja hidrolaze ILL2, što je u skladu s dosadašnjim rezultatima istraživanja. Analizirane su mutante s utišanom ekspresijom gena AtILR1, AtILL2 i AtIAR3, te je metodama PCR-a i elektroforeze izdvojena jednostruka nul mutanta ill2-1 u kojoj je identificirana insercijska mutacija u genu AtILL2 koja uzrokuje pomak u okviru čitanja i sintezu krnjeg, nefunkcionalnog proteina. Metodama PCR-a i sekvenciranja izdvojene su jednostruke mutante ilr1-1 i iar3-2 u kojima je identificirana mutacija Gly224Glu u genu AtIAR3 (mutanta iar3-2) i mutacija Gly139Asp u genu AtILR1 (mutanta ilr1-1) koje uzrokuju potpuni ili djelomični gubitak funkcije hidrolaza ILR1 i IAR3. Kombinacijom PCR-a, elektroforeze i sekvenciranja izdvojena je dvostruka mutanta ill2-1/iar3-2 koja ima mutaciju Gly224Glu u genu AtIAR3 i insercijsku mutaciju u genu AtILL2, i trostruka mutanta ilr1-1/ill2-1/iar3-2 koja ima mutaciju Gly139Asp u genu AtILR1, insercijsku mutaciju u genu AtILL2 i mutaciju Gly224Glu u genu AtIAR3. Mutante dobivene i analizirane u ovom radu dalje će se koristiti za istraživanja metabolizam IAA naročito tijekom izloženosti biljaka abiotskom stresu.

Abstract (english)

Indole acetic acid (IAA) is the most dominant form of the naturally occurring auxin in Arabidopsis thaliana. Here, the mechanisms of biosynthesis, transport and conjugation of IAA in A. thaliana are reviewed, and mutants involved in IAA-aminoacid hydrolyze analyzed and regenerated. Binary vector pHOV:AtILL2-GFP was synthesized by using in-fusion technology, and transgenic A. thaliana plants, ecotypes WS and Col-0, with overexpression of AtILL2 and AtILL2-GFP transgenes were regenerated after floral dip transformation. Localization of protein AtILL2-GFP, tracked by fluorescence microscopy, suggest endoplasmic reticulum as the site of action of hydrolase ILL2. Mutants with reduced expression of AtILR1, AtILL2 and AtIAR3 genes were analyzed. Using PCR and electrophoresis mutant ill2-1 with insertional mutation in gene AtILL2, which causes synthesis of truncated nonfunctional protein, was confirmed. Mutants ilr1-1 and iar3-2 were analysed using PCR and sequencing. The mutations Gly224Glu, and Gly139Asp were confirmed in AtIAR3 (iar3-2 mutant) and AtILR1 gene (ilr1-1 mutant), respectively. The double mutant ill2-1/iar3-2 contained mutation Gly224Glu in gene AtIAR3 and insertional mutation in gene AtILL2, and the triple mutant ilr1-1/ill2-1/iar3-2 contained mutation Gly139Asp in gene AtILR1, insertional mutation in gene AtILL2 and mutation Gly224Glu in gene AtIAR3. All mutants will be used further to investigate IAA metabolism, especially during abiotic stress.

• Keywords: indol-3-octena kiselina; konjugati indol-3-octene kiseline; pojačana ekspresija; floral dip
• Keywords (english): indole-3-acetic acid; indole-3-acetic acid conjugates; overexpression; floral dip
• Language: croatian
• URN:NBN: urn:nbn:hr:217:190534
• 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: 2017-04-20 11:17:24