doktorska disertacija
Špela Mestinšek Mubi (Avtor), Jana Murovec (Mentor)

Povzetek

Tehnologija CRISPR/Cas9 predstavlja ogromen potencial za žlahtnjenje rastlin in rastlinsko biotehnologijo, saj omogoča natančno spreminjanje nukleotidnega zaporedja endogenih genov in regulacijo njihovega izražanja. Tehnologija je najbolj uporabljena za induciranje mutacij zaradi svoje preprostosti, vsestranskosti, učinkovitosti in specifičnosti. V raziskavi smo uporabili mikrospore in protoplaste petih vrst iz rodu Brassica, z namenom razvoja metode preurejanja genomov z uporabo plazmidov in ribonukleoproteinskih kompleksov (RNP), ki so sestavljeni iz encima Cas9 in enojne vodilne molekule RNA, kateri ne vključujejo koraka stabilne transformacije rastlin. Na mikrosporah smo izvedli poskuse mikrosporne embriogeneze in regeneracije rastlin s tretiranjem celic z abscizinsko kislino in butionin sulfoksiminom za izboljšano regeneracijsko sposobnost, ter kolhicinom z namenom podvojevanja kromosomov in pridobivanja podvojenih haploidnih rastlin, katerih ploidnost smo določevali s pretočno citometrijo. Iz izoliranih protoplastov smo preverjali regeneracijsko sposobnost poganjkov. V mikrospore in protoplaste smo s polietilen glikolom (PEG) in elektroporacijo vnašali plazmide brez ali z geni za CRISPR/Cas9 ter RNP z zapisom za gen FRIGIDA. Uspešnost vnosa plazmidnih vektorjev v protoplaste smo določevali s pretočnim citometrom. Za detekcijo indel mutacij smo uporabili sekvenciranje naslednje generacije. Delež induciranih indel mutacij je pri transformaciji mikrospor s plazmidi ali RNP znašal do 0,03 %, po transformaciji protoplastov z vnosom RNP do 6,00 % in do 2,41 % pri vnosu plazmidnih vektorjev.

Ključne besede

CRISPR/Cas9;ribonukleoproteinski kompleksi;RNP;mikrospore;protoplasti;preurejanje genomov;Brassica;

Podatki

Jezik: Slovenski jezik
Leto izida:
Tipologija: 2.08 - Doktorska disertacija
Organizacija: UL BF - Biotehniška fakulteta
Založnik: [Š. Mestinšek Mubi]
UDK: 582.683:606:631.528:577.21(043.3)
COBISS: 201006851 Povezava se bo odprla v novem oknu
Št. ogledov: 68
Št. prenosov: 22
Ocena: 0 (0 glasov)
Metapodatki: JSON JSON-RDF JSON-LD TURTLE N-TRIPLES XML RDFA MICRODATA DC-XML DC-RDF RDF

Ostali podatki

Sekundarni jezik: Angleški jezik
Sekundarni naslov: Genome editing of Brassica species with Cas9 ribonucleoprotein complexes
Sekundarni povzetek: The CRISPR/Cas9 technology represents enormous potential for plant breeding and plant biotechnology, as it allows precise modification of the nucleotide sequence of endogenous genes and the regulation of their expression. The technology is most commonly used for inducing mutations due to its simplicity, versatility, efficiency, and specificity. In our research, we utilized microspores and protoplasts of five species from the Brassica genus to develop a method for genome editing using plasmids and ribonucleoprotein complexes (RNPs) composed of the Cas9 enzyme and a single guide RNA molecule, excluding the step of stable plant transformation. Experiments were conducted on microspore embryogenesis and plant regeneration by treating cells with abscisic acid and buthionine sulfoximine to enhance regenerative ability. Colchicine was used to double the chromosomes and obtain doubled haploid plants, whose ploidy was determined by flow cytometry. The regeneration ability of the shoots was checked from the isolated protoplasts. Plasmids, both with and without CRISPR/Cas9 genes, and RNPs containing the FRIGIDA gene sequence were introduced into microspores and protoplasts using polyethylene glycol (PEG) and electroporation. The efficiency of introducing plasmid vectors into protoplasts was determined by flow cytometry. Next-generation sequencing was used to detect indel mutations. The percentage of induced indel mutations in microspore transformation with plasmids or RNPs ranged up to 0.03%, reaching up to 6.00% after protoplast transformation with RNP, and up to 2.41% with plasmid vector uptake.
Sekundarne ključne besede: CRISPR/Cas9;ribonucleoprotein complexes;RNPs;microspores;protoplasts;genome editing;Brassica;
Vrsta dela (COBISS): Doktorsko delo/naloga
Študijski program: 0
Komentar na gradivo: Univ. v Ljubljani, Biotehniška fak.
Strani: 1 spletni vir (1 datoteka PDF (XIII, 110 str.))
ID: 24524880