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Identifikacija ključnih cis-regulatornih elementov promotorja gena CPI8 za odziv na jasmonsko kislino
ID Matijašić, Kristina (Author), ID Lukan, Tjaša (Mentor) More about this mentor... This link opens in a new window

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Abstract
Izpostavljenost krompirja (Solanum tuberosum L.) biotskemu stresu povzroči spremenjeno izražanje na stres odzivnih genov, preko omogočanja ali zavrtja vezave prepisovalnih dejavnikov na cis-regulatorne elemente. V magistrskem delu smo analizirali promotor gena CPI8, da bi ugotovili, kateri cis-regulatorni element ali kombinacija več teh je ključna za odziv na jasmonsko kislino (JA), ki se tvori ob mehanski poškodbi tkiva in napadu koloradskega hrošča (Leptinotarsa decemlineata). Zaporedje različice tega promotorja iz rastline krompirja genotipa Désirée, pCPI8.De7, smo z verižno reakcijo s polimerazo (PCR) skrajšali na 3’- in 5’-koncu tako, da smo iz njega postopno izločali bioinformatsko predvidene cis-regulatorne elemente, odzivne na JA. Odseke pCPI8.De7 smo vstavili pred kodirajoče zaporedje gena za reporterski protein luciferazo. Nato smo prehodno transformirali rastline krompirja in tobaka (Nicotiana benthamiana Domin) in po tretiranju z JA in dodatku luciferina merili luminiscenco. Ugotovili smo, da krompir ni primeren za primerjavo aktivnosti odsekov pCPI8.De7. Z luciferaznimi testi na rastlinah tobaka smo odkrili ključen odsek pCPI8.De7 za odziv na JA, ki smo ga nato z mutagenim PCR dodatno skrajšali in vanj vnesli točkovne mutacije. Ugotovili smo, da je za odziv na JA najverjetneje ključna kombinacija vezavnih mest za prepisovalna dejavnika VIP1 in NAC078 ter še nepoznanega cis-regulatornega elementa. Te elemente bi lahko uporabili za razvoj gensko zapisanega biosenzorja za JA, kar bi omogočilo boljše razumevanje mehanizmov obrambe na stres pri krompirju.

Language:Slovenian
Keywords:biosenzor, inhibitor cisteinskih proteinaz, jasmonska kislina, luciferazni test aktivnosti promotorjev, rastlinski odziv na stres, regulacija prepisovanja, Solanum tuberosum
Work type:Master's thesis/paper
Typology:2.09 - Master's Thesis
Organization:BF - Biotechnical Faculty
Year:2023
PID:20.500.12556/RUL-146845 This link opens in a new window
COBISS.SI-ID:156794627 This link opens in a new window
Publication date in RUL:15.06.2023
Views:792
Downloads:48
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Secondary language

Language:English
Title:Identification of the key cis-regulatory elements of the promoter of the CPI8 gene for the response to jasmonic acid
Abstract:
Exposure of potato (Solanum tuberosum L.) to biotic stress results in altered expression of genes that respond to stress by activating or deactivating binding of transcription factors to cis-regulatory elements. In this master's thesis, we analysed the CPI8 gene promoter to determine a cis-regulatory element, or a combination of several elements, crucial for the response to jasmonic acid (JA), which is synthesized during stresses such as mechanical tissue damage and Colorado potato beetle infestation (Leptinotarsa decemlineata). First, we truncated the sequence of the promoter variant from potato genotype Désirée, pCPI8.De7, on its 3’ and 5’ end using polymerase chain reaction (PCR) to gradually exclude predicted JA responsive cis-regulatory elements. We fused the truncated versions of pCPI8.De7 to the coding sequence of the reporter protein luciferase. We then performed transient transformation of potato and tobacco (Nicotiana benthamiana Domin) plants and measured luminescence after addition of luciferin and JA. Results showed that potato is not suitable for such an assay. On the other hand, using luciferase assays on tobacco, we identified a section in the CPI8.De7 promoter that is crucial for the response to JA. Using mutagenic PCR, we further truncated this section and introduced several point mutations and discovered that most likely, the combination of binding sites for the transcription factors VIP1 and NAC078 and a yet unknown cis-regulatory element is indispensable for the response of pCPI8.De7 to JA. These elements could be used to develop a genetically encoded biosensor for JA that would enable a better understanding of stress defense mechanisms in potato.

Keywords:biosensor, cysteine proteinase inhibitor, jasmonic acid, luciferase test of promoter activity, plant response to stress, transcriptional regulation, Solanum tuberosum

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