The thesis deals with the mutational analysis of conserved amino acids of Potato virus X coat protein (PVX CP). The importance of selected amino acids for the spread of the virus in the plant should be determined. Nicotiana benthamiana was selected as an experimental plant. Mutations of the PVX CP were based on the comparison of PVX CP amino acid sequence with the sequence of Papaya mosaic virus coat protein (PapMV CP), the only representative of potexvirus, which includes PVX, with the described crystal structure of the CP. The importance of certain amino acids for interaction of coat protein subunits PapMV CP,CP and PapMV CP,RNA and thus for virus particles formation was experimentally determined. The available amino acid sequences of isolates and strains of PVX CP were obtained from the National Center for Biotechnology Information (NCBI) database, compared with each other and alsowith the sequence of PVX CP used in the Laboratory of Virology, Institute of Experimental Botany of the CAS. Codons encoding conserved phenylalanine and lysine at positions 33 and 118 in the PVX CP amino acid sequence were mutated using methods of molecular biology.
Five constructs of PVX CP mutants were prepared, Two deletion mutants of the PVX CP N-terminus which were created in the vector derived from PVX (pGR106) in which the cDNA sequence of PVX is integrated, the remaining 3 point mutants were prepared only as a product of SOE PCR. The reporter gene GFP for monitoring of infection and spread of mutants in plant tissue was cloned into pGR106 carrying the deletion mutants of the N-terminus of PVX CP (deletion of 2.-32. or 2.-33. amino acid). Both mutants are able to move only in a short distance from infected cells to adjacent cells within the inoculated N. benthamiana leaf. These two deletion mutants showed difference in the speed and in the extent of the GFP signal spread. Deletion mutant still possessing the codon for F33 showed faster onset of the GFP signal and was able to spread more rapidly to surrounding cells in comparison with deletion mutant, where the codon for F33 was removed. Other mutants carrying the point mutations were also prepared: the deletion mutant of the codon for F33 (deletion F33), the substitution mutant, in which the codon for phenylalanine at position 33 was replaced by the codon for alanine (F33A) and substitution mutant, where the codon for lysine at position 118 was replaced by the codon for alanine (K118A). Unfortunately, all three point mutants could not be cloned into the vector pGR106, therefore, the evaluation of their spread in N. benthamiana plants was not possible.
Based on obtained results it is possible to conclude that the amino acid F33 is important for the intercellular movement within the neighboring cells. To assess the importance of the amino acids F33 and K118 in the systemic infection, it would be necessary to evaluate also the point mutants deletion F33, F33A, and K118.
| Identifer | oai:union.ndltd.org:nusl.cz/oai:invenio.nusl.cz:258763 |
| Date | January 2016 |
| Creators | Werschallová, Markéta |
| Contributors | Ryšánek, Pavel, Jan, Jan |
| Publisher | Česká zemědělská univerzita v Praze |
| Source Sets | Czech ETDs |
| Language | Czech |
| Detected Language | English |
| Type | info:eu-repo/semantics/masterThesis |
| Rights | info:eu-repo/semantics/restrictedAccess |
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