Overexpression of genes using stable transformation of plants is a widly used and very efficient method for functional studies of genes of interest. The constitutive gene expression can limit plant growth. When using inducible gene expression, these limitations are overcome by using a system that activates expression of a certain gene at specified time. In this master thesis we set up a glucocorticoid receptor-based inducible gene expression system, inducible by dexamethasone. In order to test this inducible system and establish the optimal plant treaments, we transiently transformed Nicotiana benthamiana plants with a plasmid in which we introduced a yellow fluorescent protein (YFP) under the control of glucocorticoid inducible promoter. We used the confocal microscopy to follow the YFP signal at certain time points on one half of the leaf amd got signal 24 h after induction. The other half was used for YFP gene expression analysis with real-time polymerase chain reaction (qPCR), where we got signal 2 h after induction. Contrary to the control plant in which the gene was not expressed. Afterwards we focused on two potato defence genes, RbohD and TGA2.1. From DNA of potato cv. Rywal we determined nucleotide sequence of two different variants of RbohD gene – RbohD1 and RbohD2. We used the glucocorticoid-inducible system for stable transformation of NahG-Rywal potato to study the function of TGA2.1 in potato defence response. By qPCR we selected two transgenic lines in which TGA2.1 gene was overexpressed. For further investigation of gene function in plant defence system we used these two lines to infect the potato plants with the potato virus Y. As the spread of PVY was limited in these lines, we confirmed the positive role of TGA2.1 in plant defence by qPCR.
|
