Analiza ekspresije exosc10/Exosc10 u tkivima zebrice (Danio rerio) i embrionalnim fazama razvoja

master's thesis

• Title (english): Analysis of exosc10/Exosc10 expression in zebrafish (Danio rerio) tissues and embryonic developmental stages
• Author: Leticija Oreški
• Mentor: Igor Stuparević (mentor)
• Granter: University of Zagreb; Faculty of Food Technology and Biotechnology; (Department of Chemistry and Biochemistry); (Laboratory for Biochemistry); Zagreb
• Defense date and country: 2019-07-01, Croatia
• Scientific / art field, discipline and subdiscipline: BIOTECHNICAL SCIENCES; Biotechnology

Abstract

Cilj ovog rada bio je provesti analizu ekspresije gena exosc10 na razini mRNA na 8 različitih tkiva odraslih ženskih i muških jedinki zebrice (Danio rerio) i 9 faza embrionalnog razvoja u različitim vremenskim intervalima nakon oplodnje, upotrebom metode PCR-a u realnom vremenu. Exosc10 egzoribonukleaza je katalitička podjedinica RNA egzosoma koji se nalazi u svim eukariotskim stanicama i ima ulogu u procesiranju, kontroli kvalitete i degradaciji RNA u jezgri i citoplazmi. Kako bi se ti rezultati povezali s razinom proteina, provedena je Western blot metoda na lizatima muških i ženskih tkiva zebrice, korištenjem 2 različita primarna antitijela za koja je predviđeno da rade na zebrici. Provedeno je i detaljno pretraživanje genomskih databaza GeneBank i Ensembl kako bi se konstruiralo filogenetsko stablo i matrica homologije. To je, uz pomoć sintenijske analize, pokazalo da za Exosc10 kod zebrice postoji ortolog kod čovjeka, da su ti protein 60% identični među kralješnjacima te da je on evolucijski konzerviran među eukariotima. exosc10 je konstitutivno i diferencijalno eksprimiran u svim testiranim tkivima, bez značajne razlike između dva spola. Gonade oba spola i ranije faze embrionalnog razvoja pokazuju najveću ekspresiju. Za gonade su rezultati ekspresije na razini proteina u korelaciji s rezultatima na razini transkripta, ali bi se analiza trebala daje optimizirati kako bi se dobila preciznija interpretacija. Oba testirana antitijela su prvi puta korištena na zebrici te su se pokazala funkcionalnim. Konačno, analiza ekspresije gena c1d, mpp6, dis3, dis3l i dis3l2 na razini mRNA je također provedena, s obzirom na to da ti geni kodiraju za podjedinice RNA egzosoma te su tako povezani sa exosc10. Ti geni su također konstitutivno i diferencijalno eksprimirani u svim testiranim tkivima, ali je primijećena značajna razlika između spolova za c1d.

Abstract (english)

The aim of this thesis was to perform exosc10 expression analysis at the level of mRNA in zebrafish (Danio rerio) using quantitative real-time PCR (qRT-PCR) with eight different tissues of both female and male fish and with embryonic developmental stages in nine different time intervals after fertilization. Exosc10 exoribonuclease is a catalytic subunit of the RNA exosome complex in eukaryotic cells and is important in processing, quality control and degradation of RNA in both the nucleus and cytoplasm of eukaryotes. To correlate this to protein expression levels, Western blot (WB) analysis was performed using both male and female zebrafish tissue lysates, using two different primary antibodies predicted to work with zebrafish. A thorough bioinformatics research of genomic databases GeneBank and Ensembl was performed to construct a phylogenetic tree and a homology matrix. All of this, along with synteny analysis, indicated that the protein is orthologous to its human counterpart, that it shares about 60% identity among vertebrates and that it is evolutionarily conserved in eukaryotes. exosc10 is constitutively and differentially expressed in all of the tissues tested, without notable difference between two genders. Gonads of both genders and earlier embryonic developmental stages show the highest expression. For gonads, results for protein expression are in correlation with qRT-PCR results, but the analysis should be optimized for further interpretation. Both antibodies used were, for the first time, proved to work on zebrafish. Lastly, gene expression analysis at the level of mRNA for c1d, mpp6, dis3, dis3l and dis3l2 was performed, since they work closely with exosc10 within the exosome. All genes are differentially and constitutively expressed in all of the tissues tested, with notable difference between two genders for c1d.

• Keywords: Exosc10 egzoribonukleaza; zebrica (Danio rerio); PCR u realnom vremenu; Western blot; bioinformatika
• Keywords (english): Exosc10 exoribonuclease; zebrafish (Danio rerio); qRT-PCR; Western blot; bioinformatics
• Language: croatian
• URN:NBN: urn:nbn:hr:159:297077
• Study programme: Title: Molecular Biotechnology; Study programme type: university; Study level: graduate; Academic / professional title: magistar/magistra inženjer/inženjerka molekularne biotehnologije (magistar/magistra inženjer/inženjerka molekularne biotehnologije)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Access restricted to students and staff of home institution
• Created on: 2019-12-06 11:01:20