Novo otkiveni i stresom inducirani proteini ASR u kulturi tkiva kaktusa Mammillaria gracilis Pfeiff.

master's thesis

• Title (english): Newly detected and stress-induced ASR proteins in cactus tissue culture Mammillaria gracilis Pfeif.
• Author: Antonio Tudić
• Mentor: Petra Peharec Štefanić (mentor)
• Committee member: Petra Peharec Štefanić (predsjednik povjerenstva)
• Committee member: Martina Šeruga Musić (član povjerenstva)
• Committee member: Željka Vidaković-Cifrek (član povjerenstva)
• Committee member: Biljana Balen (član povjerenstva)
• Granter: University of Zagreb; Faculty of Science; (Department of Biology); Zagreb
• Defense date and country: 2019-02-21, Croatia
• Scientific / art field, discipline and subdiscipline: NATURAL SCIENCES; Biology

Abstract

Proteini inducirani apscizinskom kiselinom, stresom i dozrijevanjem (ASR) su specifični za biljke, imaju malu molekulsku masu i stabilni su pri povišenoj temperaturi zbog izraţene hidrofilnosti. PronaĎeni su u jezgri i citoplazmi, što upućuje na to da se radi o transkripcijskim faktorima i šaperonima. Osim u odgovoru na sušu i salinitet, uključeni su u regulaciju metabolizma šećera, aminokiselina s razgranatim bočnim lancima i stanične stijenke. Proteini ASR nisu prisutni u svim biljkama, primjerice, geni ASR nisu pronaĎeni u modelnom organizmu Arabidopsis thaliana. TakoĎer, proteini slični proteinu ASR nisu poznati za porodicu kaktusa, koju karakterizira visoka učinkovitost korištenja vode i otpornost na sušu. Ovim istraţivanjem dokazano je, po prvi put, postojanje proteina sličnih proteinu ASR pročišćavanjem afinitetnom kromatografijom, razdvajanjem proteina jedno- i dvodimenzionalnom SDS-PAG elektroforezom te de novo sekvenciranjem peptida spektrometrijom masa. Prijenosom proteina na membranu i detekcijom specifičnim αASR1 antitijelom, uočena je promjena ekspresije proteina sličnog proteinu ASR u tkivima kaktusa (izdanak, kalus, tumor) pod utjecajem solnog i osmotskog stresa. Pokazano je da solni stres dovodi do veće ekspresije proteina sličnog proteinu ASR u odnosu na osmotski stres. Umnaţanjem cDNA degeneriranim početnicama te molekularnim kloniranjem identificirana je parcijalna cDNA gena sličnog genu ASR koja odgovara konzerviranim motivima 1 i 2 proteina ASR.

Abstract (english)

Abscisic acid-, stress-, and ripening- induced (ASR) proteins are plant specific, low molecular weight and heat-stable proteins that have pronounced hydrophilicity. They were found in the nucleus and cytoplasm, indicating their transcription factor and chaperon-like activities. They are involved not only in plant drought and salinity tolerance, but also in regulation of sugar, branched amino acids and cell wall metabolism. ASR proteins are not present in all plant species, for instance, ASR genes are not found in model organism Arabidopsis thaliana. Also, ASR-like proteins are not known for Cactaceae family, characterised by high water efficiency and drought tolerance. This research has proven, for the first time, presence of ASR-like proteins by means of affinity chromatography purification, one- and two-dimensional SDS-PAGE proteins separation and mass spectrometry de novo peptide sequencing. Using western blot and specific αASR1 antibody, changes in expression profiles of ASR-like proteins in cacti tissues (shoot, callus, tumor) affected by salt and osmotic stress was noticed. It was shown that salt stress leads to higher expression of ASR-like protein compared to the osmotic stress. cDNA amplification with degenerate primers and molecular cloning was used for identification of partial cDNA ASR-like gene corresponding to conserved motifs 1 and 2 of ASR protein.

• Keywords: ASR proteini; Cactaceae; Ni-NTA agaroza; 2D elektroforeza; de novo sekvenciranje peptida; abiotički stres; parcijalna cDNA
• Keywords (english): ASR proteins; Cactaceae; Ni-NTA agarose; 2D electrophoresis; de novo peptide sequencing; abiotic stress; partial cDNA
• Language: croatian
• URN:NBN: urn:nbn:hr:217:535146
• Study programme: Title: Molecular Biology; Study programme type: university; Study level: graduate; Academic / professional title: magistar/magistra molekularne biologije (magistar/magistra molekularne biologije)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access; Embargo expiration date: 2020-02-21
• Created on: 2019-03-26 09:19:44