20.500.12556/RUL-133385 Lokalizacija egerolizinskega proteina v nitasti glivi Aspergillus niger Localization of aegerolysin protein in filamentous fungus Aspergillus niger Genom nitaste glive Aspergillus niger vsebuje dva genska zapisa za proteina egerolizinske družine, nigerolizin A1 (NigA1) in nigerolizin A2 (NigA2). Za nigerolizina NigA1 in NigA2 se predvideva, da bi lahko sodelovala pri obrambi organizma. Do sedaj so ugotovili, da se nigerolizina izločata iz glive, saj sta bila prisotna v sekretomu. Zaradi odsotnosti signalnega peptida naj bi se izločala z nekonvencionalno sekrecijo. Z namenom ugotoviti lokacijo egerolizinov NigA1 in NigA2 na celični in/ali zunajcelični ravni smo uporabili različna javno dostopna spletna orodja za napovedovanje prisotnosti signalnih peptidov, transmembranskih domen, glikozilacijskih mest in morebitne celične in/ali zunajcelične lokacije. Z uporabo konfokalne mikroskopije, slikanjem živega micelija sevov glive Aspergillus niger in z uporabo fluorescenčnih sond in barvil smo ugotovili, da se NigA1-mCherry, NigA2-mCherry in NigA2-eYPF nahajajo pri mladih hifah, enakomerno razporejeni v citosolu celice, s staranjem hif pa se fluorescenca pojavi tudi znotraj vakuol. Prav tako sta oba egerolizina prisotna v sporah. Prisotnost NigA2 v citoplazmi hif smo potrdili še z imunooznačevanjem NigA2 s protitelesi. V primeru gojenja seva glive Aspergillus niger ob bakterijah Bacillus cereus je prišlo do morebitne interakcije med organizmoma in možnega izločanja NigA2-eYPF in NigA2-mCherry iz glive, kar kaže na morebitno vlogo NigA2 pri obrambnih mehanizmih glive. Delecija enega ali drugega gena nigerolizina bi lahko imela vpliv na zmanjšano kalitev spor glive. The genome of the filamentous fungus Aspergillus niger contains two genes for aegerolysin proteins, nigerolysin A1 (NigA1) and nigerolysin A2 (NigA2). For nigerolysins NigA1 and NigA2 suggested that they may be involved in fungal defense mechanisms. Previously, it was assumed that the nigerolysins were secreted by the fungus. Due to the lack of a signal peptide, they should be secreted by unconventional secretion. To determine the localization of the aegerolysins NigA1 and NigA2 at the cellular and/or extracellular level, we used several publicly available online tools to predict the presence of signal peptides, transmembrane domains, glycosylation sites and possible cellular and/or extracellular localization. Using confocal microscopy, imaging of live mycelium of Aspergillus niger strains and fluorescent probes and dyes, we found that NigA1-mCherry, NigA2-mCherry, and NigA2-eYFP are uniformly distributed in the cytosol of the cell when young, and fluorescence also occurs in vacuoles as hyphae age. The presence of NigA2 in the cytoplasm of hyphae was further confirmed by immunolabeling NigA2 with antibodies. Both aegerolysins are also present in spores. Cultivation of Aspergillus niger with Bacillus cereus led to a possible interaction between the organisms and possible secretion of NigA2-eYFP and NigA2-mCherry from the fungus. Deletion of one or the other nigerolysin gene could have an effect on reduced germination of fungal spores. egerolizini Aspergillus niger NigA1 NigA2 nekonvencionalna sekrecija aegerolysins Aspergillus niger NigA1 NigA2 unconventional secretion true false false Slovenski jezik Angleški jezik Magistrsko delo/naloga 2021-11-25 07:15:18 2021-11-25 07:15:30 2022-09-12 04:16:05 0000-00-00 00:00:00 2021 0 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 1970-01-01 202819 21674.pdf 21674.pdf 1 40F6EE2CC6801AF8462208F164459082 f6ec1581e21cebd134c320cddf3c75c94abd9c0ed6eb78847afcd6e9cd6a6f87 00dfbb78-4db7-11ec-abdb-00155dcfd717 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=151408 Biotehniška fakulteta 0 0 0