| Abstract [eng] |
The aim of this work was to verify the possibility to increase expression of yeast (Saccharomyces cerevisiae), K2 type killer preprotoxin in plants, using constitutive cauliflower mosaic virus CaMV 35S promoter. In order to achieve it, the following was done: selection of unoncogenic strain of agrobacterium (Agrobacterium tumefaciens) with plant transformation vector pART27-KillK2, containing K2 type killer preprotoxin gene; transformation of plain tobacco (Nicotiana tabacum L.) with mentioned agrobacteria; selection of Nicotana tabacum L. transgenic callus. Following the success of the Agrobacterium tumefaciens conjugation with E.coli strain (containing killer plasmid pART27-KillK2) and successful transformation of model N.tabacum plant with A.tumefaciens, there was used the PCR method to check out the killer effect of it. PCR and DNA electrophoresis showed that KillK2 gene is incorporated into plant genome and there is weak expression of this gene. However there was not found the promoter of couliflower mosaic virus CaMV 35S upstream this gene. In the previous studies, it was considered that the expression of mentioned transformed gene exist only when the transcription of it is directly regulated by alcoholdehidrogenase ADH1 promoter. However after investigation it was shown that promoter ADH1 isn’t connected to beginning of gene KillK2. There is the possibility that both ADH1 and CaMV35S promoters are remote from the beginning of KillK2 gene, or the iniciation is done by other regulators, which are inefective. Probably for this reason in transformed plants (with both pGA482-KillK2 and pART-KillK2 vectors) there was observed weak and constitutive expression of preprotoxin. Also the specifics of protien maturation in plants may have had effect. Killer preprotoxin maturation in plants (N.tabacum) and yeasts (S.cerevisiae) is very different because of absence or exstence of specific proteases or glycosylation. Even after successful case of transformation it is not known how the cells of plant react to such toxin formation inside or secretion outside cell. Since plant cells are very different from the yeast cells, the functiality of killer toxin may have significant influence, such as their viability. In order to fully answer these questions, there is planned more detailed study of transformed plants in the future. |
