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Indukcija mestno specifičnih mutacij z vnosom ribonukleoproteinskih kompleksov v protoplaste oljne ogrščice (Brassica napus L.)
Mazinjanin, Sabina (Author), Murovec, Jana (Mentor) More about this mentor... This link opens in a new window

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Abstract
CRISPR/Cas9 je kot orodje genskega inženirstva vse pomembnejše pri tarčnem preurejanju genomov rastlin. Neposreden vnos sgRNA in proteina Cas9 v protoplaste rastlinskih vrst s pomočjo PEG je ena izmed metod, ki omogoča mutagenezo genoma brez transgeneze. V protoplaste oljne ogrščice (Brassica napus L.) smo želeli s pomočjo PEG vnesti ribonukleoproteinske komplekse in s tem inducirati tarčne mutacije brez genske transformacije rastlin. V ta namen smo optimizirali pripravo rastlinskega materiala, sestavo encimske raztopine za razgradnjo celične stene ter sam postopek izolacije protoplastov. Ugotovili smo, da so kot izhodni material najprimernejši etiolirani hipokotili, za ustrezno razgradnjo celične stene pa sta se za najprimernejši izkazali celulazi Onozuka R-10 in Onozuka RS. Najprimernejši postopek izolacije smo izbrali na podlagi živosti in koncentracije protoplastov ter uspešnosti vnosa ekspresijskega plazmida z genom ZsGreen. Za tarčno mutagenezo protoplastov smo uporabili štiri različne sgRNA, s tarčami znotraj gena pds, ki smo jih v kompleksu z encimom Cas9 vnašali s pomočjo PEG. Po 48 urni inkubaciji smo iz protoplastov izolirali DNA in s pomočjo endonukleaze T7, RFLP analize ter z metodo sekvenciranja po Sangerju skušali identificirati morebitne tarčne mutacije. Rezultati encimskih testov so nakazovali uspešno indukcijo tarčnih mutacij, vendar jih s sekvenčno analizo po Sangerju nismo mogli z gotovostjo potrditi. Za izboljšanje rezultatov bi bila potrebna nadaljnja optimizacija postopka izolacije protoplastov in/ali transformacije ter uporaba občutljivejše metode identifikacije mutacij.

Language:Slovenian
Keywords:oljna ogrščica, protoplasti, transformacija, PEG, tarčna mutageneza, CRISPR/Cas9
Work type:Master's thesis/paper (mb22)
Tipology:2.09 - Master's Thesis
Organization:BF - Biotechnical Faculty
Year:2017
Publisher:[S. Mazinjanin]
UDC:633.853.492:631.528.1:601.4:575.224.4(043.2)
COBISS.SI-ID:8801145 Link is opened in a new window
Views:339
Downloads:186
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Secondary language

Language:English
Title:Induction of site-specific mutations with ribonucleoprotein complexes in rapeseed (Brassica napus L.) protoplasts
Abstract:
CRISPR/Cas9 is gaining importance as a gene engineering tool for targeted gene modifications of plant genomes. Use of PEG-stimulated sgRNA and Cas9 protein direct uptake into protoplasts is one of the methods that enable transgene-free targeted mutagenesis. The aim of our work was to deliver ribonucleoprotein complexes into rapeseed (Brassica napus L.) protoplasts using PEG and in this manner induce target specific mutations. For this purpose we optimized preparation of plant material, composition of enzyme solution for degradation of cell walls and the procedure of protoplasts isolation. We found that etiolated hypocotyls are the most suitable plant material and that cellulases Onozuka R-10 and Onozuka RS are the most effective for degradation of cell walls. The most appropriate isolation protocol was chosen based on protoplasts viability and concentration and also on successful insertion of expression plasmid containing reporter gene ZsGreen. For targeted mutagenesis we used four different sgRNAs with targets in pds gene, in complex with Cas9 enzyme. After 48 hours of incubation we isolated DNA from protoplasts. To identify possible target mutations we used T7 endonuclease assay, RFLP analysis and Sanger sequencing. The results of enzyme tests indicated successful induction of targeted mutations, but could not be reliably verified with Sanger sequencing. In order to obtain better results further optimization of protoplast isolation and/or transformation procces is needed and also the use of more sensitive methods for identification of targeted mutations.

Keywords:rapeseed, protoplasts, transformation, PEG, targeted mutagenesis, CRISPR/Cas9

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