Vztah mezi metylací DNA a expresí rostlinných transgenů v systému Nicotiana tabacum

Mgr. Lucie Khaitová, Ph.D.

Master's thesis

Relationship between DNA methylation and expression of plant transgenes in the system Nicotiana tabacum

Abstract:

English annotation This work was focused on the study of the in trans silencing capacity of the trigger locus 271. This locus has been shown to induce inactivation and methylation of the 35S and 19S viral promoters in plant transgenic systems. Locus 271 was combined with the transgenic acceptor loci carrying the bacterial neomycin phosphotransferase II reporter gene driven by the 35S promoter in tobacco hybrid plants. Two genomic organizations of the acceptor loci can be recognized: (i) a single T-DNA insertion within the locus 2 actively expressing the reporter transgene (ii) inverted repeat configuration of two T-DNA copies residing locus 1. The nptII genes in this locus are posttranscriptionally silenced (PTGS). In 271/1 and 271/2 hybrids, we determined expression of the nptII reporter transgene. In parallel, methylation of promoter sequences was analyzed. The silencer locus 271 induced significant reduction of the nptII expression in both locus 1 and locus 2. However, while decrease of RNA levels was readily detectable in 271/2 hybrids by conventional Northern blot analysis we were unable to demonstrate silencing effect in 271/1 line due to low nptII-RNA levels in parental HeLo1 line. To increase the sensitivity of expression analysis we introduced Q-PCR method. Indeed, using this method we managed to show strong silencing effect even in 271/1 line. In 271/1 hybrids the nptII expression levels were beyond sensitivity limit of Q-PCR suggesting that PTGS has been switched into TGS. In this context, Q-PCR method seems to be more suitable for elucidation of the subtle differences in gene expression status. The presence of the silencer locus led to in trans methylation of the 35S promoter regions of both acceptor loci, although the intensity of methylation changes was different. In locus 1, which is originally methylated in the nptII coding region, methylation of cytosines located in both symmetrical and non-symmetrical sequence contexts was detected; non-methylated locus 2 acquired methylation of non-symmetrically localized cytosines only. Considering known correlation between TGS and methylation of promoter region, it is likely that 271/1 line displayed stronger TGS phenotype than the 271/2 line. Perhaps, the pre-existed methylation influenced the rate and intensity of in trans-induced methylation changes. Alternatively inverted repeat configuration could acquire unusual chromatin structure(s) highly prone to silencing effects. This project will continue by addressing the following questions: (i) what is the distribution of methylation of the promoter region in the further generations of 271/1 and 271/2 hybrids? (ii) what will be expression and methylation patterns following segregation of the silencing trigger?

Keywords

Epigenetics, Transcriptional silencing, posttranscriptional silencing, DNA methylation, transgene, in trans silencing

Language used: English

Date on which the thesis was submitted / produced: 22. 5. 2007

Thesis defence

• Date of defence: 7. 6. 2007
• Supervisor: RNDr. Aleš Kovařík, CSc.

Masaryk University

Faculty of Science

Master programme / field:
Chemistry / Biochemistry