Testing of candidate genes for compound biosynthesis in Arnica montana

master's thesis

• Title (croatian): Testiranje gena kandidata uključenih u biosintezu metabolita biljke Arnica montana
• Author: Nikolina Mrakovčić
• Mentor: Gundula Noll (mentor)
• Mentor: Nataša Bauer (sumentor)
• Committee member: Nataša Bauer (predsjednik povjerenstva)
• Committee member: Mirta Tkalec (član povjerenstva)
• Committee member: Biljana Balen (član povjerenstva)
• Committee member: Marin Ježić (član povjerenstva)
• Granter: University of Zagreb; Faculty of Science; (Department of Biology); Zagreb
• Defense date and country: 2022-02-28, Croatia
• Scientific / art field, discipline and subdiscipline: NATURAL SCIENCES; Biology

Abstract

Arnica montana L. is a traditional medicinal plant used for centuries. It owes its medicinal
properties to sesquiterpene lactones, helenalin and dihydrohelenalin. Traditionally, the plants
are harvested from its natural habitat, but there is need for alternative ways to sustainably
obtain bioactive compounds. To achieve alternative production of A. montana bioactive
sesquiterpene lactones, their biosynthesis pathway must be elucidated. Functional
characterization of candidate genes, that might be involved in sesquiterpene lactones
biosynthesis, has been conducted in the heterologous expression system yeast Saccharomyces
cerevisiae and compounds analysis via gas chromatography-mass spectrometry. Additionally,
analysis of potential candidate genes expression patterns and sesquiterpene lactones content
distribution in plants was carried out. Candidate genes AmGAS and AmGAO were verified to
encode respective germacrene A synthase and germacrene A acid oxidase, and their enzymatic
activity was confirmed. Candidate genes AmCYPs were functionally analysed to a certain extent,
with candidate AmCYP7 showing great potential for further investigation. Correlation of
expression patterns and content of bioactive compounds in A. montana tissues revealed that
among tested, AmCYP7 gene has potential to be part of sesquiterpene lactones biosynthesis
pathway and provided preliminary evidence for their biosynthesis at different developmental
stages during the plant´s life cycle.

Abstract (croatian)

Arnica montana L. je biljka koja se stoljećima koristi u tradicionalnoj medicini. Za njezina
ljekovita svojstva odgovorni su seskviterpenski laktoni, helenalin i dihidrohelenalin. Arnika se
tradicionalno ubire s prirodnog staništa i postoji potreba za alternativnim i održivijim načinima
sinteze bioaktivnih sastojaka. Kako bi alternativna proizvodnja bioaktivnih seskviterpenskih
laktona bila moguća, potrebno je razjasniti put biosinteze. Provedena je funkcionalna
karakterizacija gena kandidata iz A. montana, koji su potencijalno uključeni u biosintetski put
seskviterpenskih laktona, u heterolognom ekspresijskom sustavu, kvascu Saccharomyces
cerevisiae. Metaboliti sintetizirani u kvascu analizirani su plinskom kromatografijom i masenom
spektrometrijom. Također, analizirani su ekspresijski obrasci gena kandidata te distribucija i
sadržaj seskviterpenskih laktona u različitim tkivima biljke A. montana. Potvrđeno je da geni
kandidati AmGAS i AmGAO kodiraju sintazu germakrina A, odnosno oksidazu germakrina A te
je potvrđena njihova enzimska aktivnost. Geni AmCYP su djelomično funkcionalno
okarakterizirani, a gen AmCYP7 pokazao je najveći potencijal za daljnje istraživanje. Korelacija
ekspresijskih obrazaca gena kandidata i sadržaja bioaktivnih sastojaka u tkivima biljke A.
montana pokazala je da je gen AmCYP7 vjerojatno uključen u biosintezu seskviterpenskih
laktona i pružila preliminarne dokaze za biosintezu seskviterpenskih laktona u različitim
razvojnim stadijima biljke A. montana.

• Keywords: helenalin; sesquiterpene lactones; biosynthesis pathways; heterologous expression; Saccharomyces cereviasiae; germacrene A; germacrene A acid
• Keywords (croatian): helenalin; seskviterpenski laktoni; biosintetski put; heterologna ekspresija; Saccharomyces cereviasiae; germakrin A; germakrinska A kiselina
• Language: english
• URN:NBN: urn:nbn:hr:217:279243
• 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: Embargoed access; Embargo expiration date: 2024-02-28
• Created on: 2022-03-16 10:21:08