magistrsko delo
Karmen Pogačar (Author), Nataša Štajner (Reviewer), Kristina Gruden (Mentor), Kristina Gruden (Thesis defence commission member), Anna Coll Rius (Thesis defence commission member), Mojca Narat (Thesis defence commission member), Nataša Štajner (Thesis defence commission member), Anna Coll Rius (Co-mentor)

Abstract

Pri genski modifikaciji rastlin se najpogosteje uporablja konstitutivno izražanje genskih konstruktov. Konstitutivno izražanje genskih konstruktov pogosto omejuje rast rastline. Pri uporabi inducibilnega izražanja se lahko izognemo tem omejitvam, saj s pomočjo inducibilnega sredstva sprožimo prepis želenega gena ob točno določenem času. V magistrskem delu smo uporabili glukokortikoidni inducibilni sistem, ki ga inducira deksametazon. Za preverjanje delovanja sistema smo rastline Nicotiana benthamiana transformirali s plazmidom, v katerega smo vstavili gen za rumeni fluorescentni protein (YFP), čigar izražanje je pod vplivom glukokortikoidnega inducibilnega promotorja. S konfokalno mikroskopijo smo v določenih časovnih točkah spremljali fluorescenco YFP v povrhnjici polovice lista in zaznali signal 24 ur po indukciji. Drugo polovico lista smo uporabili za preverjanje izražanja gena YFP z verižno reakcijo s polimerazo v realnem času (qPCR) in zaznali signal 2 uri po indukciji. Pri kontrolni rastlini signala nismo zaznali. V naslednjem koraku smo se osredotočili na dva obrambna gena krompirja, RbohD in TGA2.1. Iz DNK krompirja sorte Rywal smo pridobili nukleotidno zaporedje dveh različic gena RbohD, in sicer RbohD1 in RbohD2. Nato smo vzpostavljeni inducibilni sistem uporabili pri stabilni transformaciji krompirja sorte NahG-Rywal za izražanje gena TGA2.1. S qPCR smo izbrali dve transgeni liniji, pri katerih je bilo po tretiranju izražanje gena TGA2.1 povečano. Uporabili smo ju za nadaljnje preučevanje funkcije gena TGA2.1 z okuževanjem rastline s krompirjevim virusom Y (PVY) in ugotovili, da povečano izražanje gena TGA2.1 pripomore k obrambi pred virusom.

Keywords

biotehnologija;glukokortikoidni inducibilni sistem;TGA2.1;RbohD;deksametazon;krompir;Solanum tuberosum;Nicotiana benthamiana;konfokalna mikroskopija;qPCR;PVY;

Data

Language: Slovenian
Year of publishing:
Typology: 2.09 - Master's Thesis
Organization: UL BF - Biotechnical Faculty
Publisher: [K. Pogačar]
UDC: 602.6:582.930.11/.3(043.2)
COBISS: 9347705 Link will open in a new window
Views: 870
Downloads: 202
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Other data

Secondary language: English
Secondary title: Establishing a system for inducible gene expression in tobacco and potato plants
Secondary abstract: Overexpression of genes using stable transformation of plants is a widly used and very efficient method for functional studies of genes of interest. The constitutive gene expression can limit plant growth. When using inducible gene expression, these limitations are overcome by using a system that activates expression of a certain gene at specified time. In this master thesis we set up a glucocorticoid receptor-based inducible gene expression system, inducible by dexamethasone. In order to test this inducible system and establish the optimal plant treaments, we transiently transformed Nicotiana benthamiana plants with a plasmid in which we introduced a yellow fluorescent protein (YFP) under the control of glucocorticoid inducible promoter. We used the confocal microscopy to follow the YFP signal at certain time points on one half of the leaf amd got signal 24 h after induction. The other half was used for YFP gene expression analysis with real-time polymerase chain reaction (qPCR), where we got signal 2 h after induction. Contrary to the control plant in which the gene was not expressed. Afterwards we focused on two potato defence genes, RbohD and TGA2.1. From DNA of potato cv. Rywal we determined nucleotide sequence of two different variants of RbohD gene – RbohD1 and RbohD2. We used the glucocorticoid-inducible system for stable transformation of NahG-Rywal potato to study the function of TGA2.1 in potato defence response. By qPCR we selected two transgenic lines in which TGA2.1 gene was overexpressed. For further investigation of gene function in plant defence system we used these two lines to infect the potato plants with the potato virus Y. As the spread of PVY was limited in these lines, we confirmed the positive role of TGA2.1 in plant defence by qPCR.
Secondary keywords: biotechnology;glucocorticoid inducible system;dexamethasone;potato;confocal microscopy;
Type (COBISS): Master's thesis/paper
Study programme: 0
Embargo end date (OpenAIRE): 1970-01-01
Thesis comment: Univ. v Ljubljani, Biotehniška fak., Študij biotehnologije
Pages: XII, 80 f., [10] f. pril.
ID: 11227547