diplomsko delo
Zarja Weingerl (Author), Ajda Taler-Verčič (Mentor)

Abstract

Jod igra pomembno vlogo v številnih organizmih. Je esencialno mikro-hranilo za ljudi in živali, njegova vloga v rastlinah pa je do danes še dokaj neraziskana. Njegovo pomankanje pri ljudeh lahko povzroča številne zdravstvene težave, zato si znanstveniki prizadevajo najti način, kako bi lahko zagotovili zadosten vnos joda v telo. Zelo potencialna je biofortifikacija pridelkov, vendar je za njeno učinkovito delovanje ključnega pomena razumevanje vloge joda v rastlinah. Ena izmed neznank so tudi jodirani proteini, ki so jih znanstveniki identificirali v listih in koreninah rastline A. thaliana (Navadni repnjakovec). Mednje sodita proteina ribuloza bisfosfat karboksilaza/oksigenaza aktivaza (RuBisCO aktivaza) in fruktoza-bisfosfat aldolaza 2 (AtFBA2), ki igrata pomembno vlogo v Calvinovem ciklu in sta ključna za različne fotosintetske organizme. Z željo po odkritju vloge joda v RuBisCO aktivazi in AtFBA2 smo se lotili molekulskega kloniranja in priprave rekombinantnih proteinov RuBisCO aktivaza in AtFBA2. Uspeli smo pripraviti zapisa za oba izbrana proteina, jima dodali N-končno heksahistidinsko oznako in prepoznavno mesto za TEV proteazo, ter ju prenesli v vektor pET-32b(+). Uspešno smo izvedli transformacijo celic E. coli seva DH5α, čemur je sledila izolacija plazmida in njegova vstavitev v ekspresijski sev. Izražanje in izolacijo obeh proteinov z Ni-afinitetno kromatografijo smo analizirali z NaDS PAGE.. Pri izražanju žal nismo bili uspešni, zato nismo mogli nadaljevati z preučevanjem funkcije joda v izbranih proteinih. Postopek, ki smo ga uporabili bo tako potrebno še nadalje optimizirati.

Keywords

jod;pomen joda;RuBisCO aktivaza;ribuloza bisfosfat karboksilaza/oksigenaza aktivaza;fruktoza-bisfosfat aldolaza 2;AtFBA2;diplomska dela;

Data

Language: Slovenian
Year of publishing:
Typology: 2.11 - Undergraduate Thesis
Organization: UL FKKT - Faculty of Chemistry and Chemical Technology
Publisher: [Z. Weingerl]
UDC: 577.15:602.7(043.2)
COBISS: 202141955 Link will open in a new window
Views: 61
Downloads: 13
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Other data

Secondary language: English
Secondary title: Cloning and expression of chloroplast proteins from A. thaliana in a bacterial expression system
Secondary abstract: Iodine plays a key role in many organisms. It is essential micro-nutrient for humans and animals, but its role in plants remains relatively unknown. Iodine deficiency in humans can cause numerous health issues, hence scientists are striving to find ways to limit its impact. Biofortification of crops is very promising, but understanding the role of iodine in plants is crucial for its effective implementation. One of the unresearched areas are iodinated proteins identified in the leaves and roots of A. thaliana. These include Ribulose-1,5-bisphosphate carboxylase/oxygenase activase (RuBisCO activase) and Fructose-bisphosphate aldolase 2 (AtFBA2), both playing important roles in the Calvin cycle and are essential for various photosynthetic organisms. With the aim of discovering the role of iodine in RuBisCO activase and AtFBA2, we begun the process of molecular cloning and preparation of recombinant proteins. We successfully prepared the sequences for both selected proteins. After the addition of N-terminal hexahistidine tag and a recognition site for TEV protease, we transferred them into the pET-32b(+) vector. We successfully transformed E. coli DH5α cells, followed by plasmid isolation and its insertion into expression strain. We continued with expression and isolation using Ni-affinity chromatography followed by the gel application and analysis of the obtained results. Because of our unsuccessful expression, we could not proceed with studying the function of iodine in the selected proteins. Thus, the process we used will need further optimization.
Secondary keywords: iodination;RuBisCO activase;AtFBA2;molecular cloning;Molekularno kloniranje;Jod;Encimi;Univerzitetna in visokošolska dela;
Type (COBISS): Bachelor thesis/paper
Study programme: 1000371
Embargo end date (OpenAIRE): 1970-01-01
Thesis comment: Univ. v Ljubljani, Fak. za kemijo in kemijsko tehnologijo, UNI Biokemija
Pages: 1 spletni vir (1 datoteka PDF (42 str.))
ID: 24373240