Analisi teknika genomiko, proteomiko eta zelularrak harizpi itxurako onddoen andui errekonbinanteak sortu eta aztertzeko

Abstract

LABURPENA: Gradu Amaierako Lan (GrAL) honen helburua biologia molekularreko eta konputazionaleko oinarrizko hainbat teknika ulertu eta lantzea izan da, metodo esperimental eta prozesu bioinformatikoak jorratuz. Horretarako, Aspergillus nidulans onddoa erabili da erreferentzia sistema bezala, bere manipulazio genetikoa prozedura estandarren arabera egin baitaiteke modu errazean eta epe motzean, gainera patogenoa izan gabe. Zehazki, KapA α-inportina proteinaren, zeinak kargo proteinen garraioa ahalbidetzen duen zitoplasmatik nukleora, eta FlbB transkripzio faktorearen, zeinak garapen asexuala kontrolatzen duen, arteko harreman hipotetiko bat aztertu nahi izan da GrAL honetan. Abiapuntua α-inportinaren bi mutante termosentikor izan dira. Termino honek giro-tenperaturan funtzionalak diren formak direla esan nahi du, baina 37 ºC-tan, tenperatura errestriktiboan, funtzionaltasuna galtzen dute. Mutazio hauek 37 ºC-tan FlbB-ren zelula barneko lokalizazio karakteristikoa aldatzen duten aztertu nahi izan da. Horretarako, analisi fenotipikoak, onddo laginen kulturarako prozedurak, DNA erauzketa prozesuak, zelulen transformazioa eta PCR erreakzioak burutu dira. Tresna bioinformatikoak ere aplikatu dira α-inportinaren mutazioek bere egitura hirudimentsionalean izan lezaketen eragina aztertzeko. Emaitzei erreparatuz baieztatu daiteke bi proteinen artean harreman genetiko bat dagoela, GFP::FlbB eta KapA::mRFP kimeren forma basatiak adierazten dituzten anduiek espora asexualen ekoizpenean galera nabaria erakusten baitute. Harreman hori molekularki baieztatzeko aukerarik balego, FlbB-ren zitoplasmatik nukleorako garraioa noiz eta nola ematen den ulertzen lagunduko luke, dagokien geneen adierazpena aktibatu edo inhibitzeko gaitasuna emanik.

ABSTRACT: The main objective of this end-of-degree project has been the understanding and training in molecular and computational biology tools. With this aim, the fungus Aspergillus nidulans has been used as the reference system, because procedures for its genetic manipulation are standard and highly accessible at the lab scale, and besides, it is not a pathogen. In this project, a hypothetical relationship between importin-α, which transports cargo proteins from the cytoplasm to nuclei, and the transcription factor FlbB, which controls asexual development, has been analysed. Two thermo-sensitive mutants of the importin-α homologue, KapA, have been used as the starting point. Thermosensitivity means that KapA is functional at 30 ºC but not at the restrictive temperature of 37 ºC. Any possible variation in the subcellular localization of FlbB at 37 ºC in these mutant genetic backgrounds has been analysed. That implied carrying out phenotypic analyses, the application of cell-culture procedures, DNA extraction, cell-transformations and multiple PCR reactions. Bioinformatics tools have also been applied to assess the effect that the corresponding importin-α mutations could have on its three-dimensional structure. Results suggest that there is a genetic relationship between both genes, since strains simultaneously expressing GFP::FlbB and KapA::mRFP chimeras show a decreased production of asexual spores. If that hypothetic relationship could be shown by molecular tools, that would render important information about when and how occurs the import of FlbB from the cytoplasm into the nucleus, in order to induce or repress the expression of target genes.