magistrsko delo
Jana Pregelj (Author), Jana Murovec (Mentor)

Abstract

V nalogi smo potrdili, da sta mogoča prenos in stabilna transformacija genov Cas9, Hpt II in ZsGreen v rastlino Nicotiana benthamiana, ki je vse bolj pomembna kot modelni organizem. Gensko transformacijo smo izvedli s pomočjo bakterije Agrobacterium tumefaciens, s katero smo kokultivirali listne izsečke in katera je omogočila vnos izbranih genov v rastlino. Adventivno regeneracijo transgenih poganjkov smo izvedli na trdnem gojišču Murashige in Skoog, ki smo mu dodali 0,1 mg l-1 avksina 1-naftalenocetne kisline (NAA), 1 mg l-1 citokinina 6-benzilaminopurina (BAP), 30 g l-1 saharoze, 150 mg l-1 timentina, 25 mg l-1 higromicina in 8 g l-1 agarja. Uspešnost transformacije smo v T0 generaciji preverili s PCR pomnoževanjem in agarozno gelsko elektroforezo, ki je sicer pokazala uspešnost prenosa genov, a je poleg tega pokazala tudi na prisotnost ostankov bakterije Agrobacterium tumefaciens. Zato smo se odločili za aklimatizacijo vseh rastlin, pri katerih so bili prisotni želeni geni, kljub prisotnosti vir genov. Stabilno transformacijo smo potrdili v T1 generaciji, s pomočjo katere smo potrdili prisotnost želenih genov in njihovo dedovanje v naslednjo generacijo. Molekulsko analizo smo v tem primeru izvedli s pomočjo KAPA3G Plant PCR kit, ki nam je omogočil hitrejše in boljše rezultate, saj je močno skrajšal pot od rastlinskega vzorca do rezultata.

Keywords

tobak;Nicotiana benthamiana;genska transformacija;Cas9;

Data

Language: Slovenian
Year of publishing:
Typology: 2.09 - Master's Thesis
Organization: UL BF - Biotechnical Faculty
Publisher: [J. Pregelj]
UDC: 577.2:601.4:633.71(043.2)
COBISS: 8799097 Link will open in a new window
Views: 1069
Downloads: 478
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Other data

Secondary language: English
Secondary title: Genetic transformation of Nicotiana benthamiana Domin.
Secondary abstract: The thesis has confirmed transmission and steady genetic transformation Cas9, Hpt II and ZsGreen into the plant Nicotiana benthamiana, which is gaining on the importance as a model organism. The gene transformation was carried out with the aid of a bacterium Agrobacterium tumefaciens which helped us to co-cultivate the leafy parts and which enabled import of the chosen genes into a plant. Adventitious regeneration of the transgenetic offshoot was carried out on a solid nutrient medium Murashige and Skoog with added 0.1 mg l-1 auxin 1-Naphthaleneacetic acid (NAA), 1 mg l-1 cytokinin 6- Benzylaminopurine (BAP), 30 g l-1 sucrose, 150 mg l-1 timentin, 25 mg l-1 higromicin and 8 g l-1 agar. The success of the transformation has been checked with PCR multiplication and agarose gel electrophoresis in T0 generation. It has proven the success of the tranmission of the genes, however, the presence of a bacterium Agrobacterium tumefaciens has also been found in traces. Therefore, the decision was made to acclimatize all the plants where the desired genes were present despite the source genes. A steady transformation has been confirmed in the T1 generation, which helped to confirm the presence of the desired genes and their inheritance in the next generation. Molecular analysis was performed with the help of the KAPA3G Plant PCR kit, which enabled faster and better results as it significantly shortened the way from a plant sample to the result.
Secondary keywords: tobacco;genetic transformation;
Type (COBISS): Master's thesis/paper
Study programme: 0
Thesis comment: Univ. v Ljubljani, Biotehniška fak., Oddelek za agronomijo
Pages: XI, 37 f., [2] f. pril.
ID: 10910896