diplomsko delo
Amadeja Volarič (Author), Zlata Luthar (Mentor)

Abstract

V predhodno pripravljene listne izsečke tobaka (Nicotiana tabacum L.) smo s pomočjo seva LBA4404 bakterije Agrobacterium tumefaciens (A. t.), ki je imela vključen plazmid pART27 2mgfp5 ER ali pCAMBIA1390 DsRed, transformirali dva markerska in dva selekcijska gena. Fenotipsko izražanje obeh markerskih transgenov gfp oz. DsRed smo spremljali v živih celicah tobaka, ki so imele sintetiziran zeleno oz. rdeče fluorescentni protein. Za ločitev transformiranih celic in tkiv od netransformiranih smo uporabili selekcijska transgena nptII in hptII odpornost na antibiotik kanamicin oz. higromicin. Po okužbi izsečkov z A. t. in plazmidom pART27 2mgfp5 ER je bila uspešnost regeneracije 82,65 % in po okužbi z genskim konstruktom A. t. pCAMBIA1390 DsRed 77,89 %. Med vitalne regenerante smo šteli tiste, ki so imeli korenine in pokončne liste. Takih je bilo na gojišču s kanamicinom 63 oz. 77,78 %. Ti so bili tudi pokazatelji uspešnosti transforamcije z nptII transgenom. Po transformaciji izsečkov z A. t. pCAMBIA1390 DsRed je nastalo 51 vitalnih regenerantov. Ti regeneranti so bili sposobni razgrajevati aktivno sestavino higromicina. Uspešnost transformacije je bila 68,92 % za hptII transgen. Pri 4 regenerantih oz. 10 % se niso namnožili fragmenti dolžine 211 bp značilni za DsRed transgen, kljub temu da smo pri njih zasledili prisotnost rdeče fluorescentnega proteina.

Keywords

tobak;Nicotiana tabacum;transformacija;Agrobacterium tumefaciens;DNA;transgen;fenotipska analiza;molekulska analiza;

Data

Language: Slovenian
Year of publishing:
Typology: 2.11 - Undergraduate Thesis
Organization: UL BF - Biotechnical Faculty
Publisher: [A. Volarič]
UDC: 602.6:582.930.3:663.9:601.4:577.21(043.2)
COBISS: 68253187 Link will open in a new window
Views: 438
Downloads: 45
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Other data

Secondary language: English
Secondary title: DNA analysis of tobacco (Nicotiana tabacum L.) after transformation by Agrobacterium tumefaciens
Secondary abstract: Two marker and two selection genes were transformed into pre-prepared tobacco leaf explants (Nicotiana tabacum L.) by Agrobacterium tumefaciens (A. t.) strain LBA4404, which included plasmid pART27 2mgfp5 ER or pCAMBIA1390 DsRed. Phenotypic expression of gfp or DsRed marker transgenes was studied in live tobacco cells that had synthesized green or red fluorescent protein. To separate the transformed cells and tissues from the non-transformed ones, we used the selection transgenes nptII and hptII resistance to the antibiotic kanamycin or hygromycin. After infection of the explants by A. t. and plasmid pART27 2mgfp5 ER, the regeneration efficiency was 82.65 % and after infection with the A. t. pCAMBIA1390 DsRed 77.89 %. Among the vital regenerants we considered those that had roots and upright leaves. There were 63 (77.78 %) on kanamycin medium. These were also indicators of the success of nptII transgene transformation. After transformation of the explants by A. t. pCAMBIA1390 DsRed was formed 51 viable regenerants. These regenerants were able to degrade the active ingredient hygromycin. The transformation success rate was 68.92 % for the hptII transgene. At 4 regenerants (10 %) fragments of 211 bp length characteristic of the DsRed transgene did not amplify, despite the presence of a red fluorescent protein.
Secondary keywords: tobacco;transformation;transgene;fenotipe analysis;molecular analysis;
Type (COBISS): Bachelor thesis/paper
Study programme: 0
Embargo end date (OpenAIRE): 1970-01-01
Thesis comment: Univ. v Ljubljani, Biotehniška fak., Oddelek za agronomijo
Pages: X, 38 f.
ID: 13089001