Izražanje nekaterih genov, vpletenih v transport kalija in natrija glive Aureobasidium pullulans, v kvasovki Saccharomyces cerevisiae
| Autor: | Markuš, Tadej |
|---|---|
| Přispěvatelé: | Gunde Cimerman, Nina |
| Jazyk: | slovinština |
| Rok vydání: | 2017 |
| Předmět: |
udc:579.25:602.6:582.28
Aureobasidium pullulans transformacija homologna rekombinacija molekularne tehnike transformation glive halotolerant fungi gene cloning halotolerantne glive protein sequence analysis homologous recombination HAK Aureobasidium pullulans ACU1 ACU2 NHA fungi molecular techniques kloniranje genov analiza proteinskih zaporedij |
| Popis: | Iz genske knjižnice poliekstremotolerantne črne kvasovke A. pullulans smo izbrali 4 različne gene – ACU1, ACU2, HAK in NHA, ki smo jih klonirali v celice S. cerevisiae s homologno rekombinacijo. Kot vektor smo uporabili plazmid pBEVY-U, ki ima kot selekcijski marker gen URA3. Plazmid smo imeli pomnožen v dveh delih, ki sta se prekrivala v selekcijskem markerju. Ob uspešni transformaciji so se fragmenti znotraj celice sestavili in selekcijski marker se je lahko izražal. Uspešne transformante smo nadaljnje testirali na gojiščih z različnimi stresnimi dejavniki. Rezultati so pokazali, da ATPaza P-tipa Acu1 omogoči rast pri visoki koncentraciji K+, črpalka Acu2 pa omogoči transformiranim celicam rast na gojiščih brez K+ ionov. Antiporterji Nha so transformiranim celicam omogočili rast na gojišču, ki je vsebovalo NaCl v koncentraciji 2 mol/L in transporterji Hak so izboljšali rast transformant na gojiščih s povišanimi koncentracijami glicerola (25 %) in LiCl (590 mM). Izvedli smo tudi in silico analize proteinskih zaporedij in rezultate primerjali z rezultati podobnih proteinov, ki smo jih dobili v bazah podatkov preko spleta. We selected four different genes from the gene library of plyextremotolerant black yeast A. pullulans and cloned them into cells of S. cerevisiae by homologous recombination. As a vector, we used plasmid pBEVY-U which has gene URA3 selection marker. The plasmid was multiplied in two parts with PCR so that they overlapped in the selection marker gene. In the case of successful transformation, fragments assembled within a cell and produced functional selection marker which could be expressed. After the transformation process we tested successfully transformed cells with few different stress factors – KCl, NaCl, LiCl, sorbitol and glycerol. The results showed that the Acu1 P-type ATPase improved growth of transformed cells in high K+ medium, whereas Acu2 ATPase allowed transformed cells to grow on the agar media with drastically reduced K+ content. Nha antiporters successfully improved yeast tolerance to NaCl, which was able to grow on medium containing NaCl at concentrations of 2 mol/L. Hak transporter have improved growth of transformed cells on medium containing up to 25 % glycerol and at high (590 mM) concentrations of LiCl. We have also performed some in silico analyses of protein sequences to gather more data about the chosen proteins. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|