Browsing by Subject "Ubiquitin-mediated proteolysis"

Ubiquitin-mediated proteolysis regulates many basic cellular processes in plant development. Especially, in Arabidopsis thaliana over 1400 genes encode components of the ubiquitin/26S proteasome pathway. Approximately, 90% of the genes encode subunits of the E3 ubiquitin ligases, which confer substrate specificity. However, till now few E3 target proteins were known. Hence, identification and characterization of E3-substrate interaction is essential for understanding the role of ubiquitinylation in various plant developmental pathways. The main objectives of my Master’s thesis were derived from the Flower- specific Ubiquitin Proteasome System (FUPS) research project on the identification of proteins related to FUPS by the candidate and genomic approaches. The candidate approach involved molecular cloning of candidate ubiquitin E3 ligase gene RFI2 and its interaction substrates SLK1 and SLK2 (SLKs). The genomic approach includes genotypic and phenotypic characterization of the Salk T-DNA mutant lines corresponding to the selected FUPS E3 components. The cloning of candidate genes RFI2 and SLKs were done by Gateway cloning technology in order to generate overexpression and inducible expression gene constructs. For functional characterization of candidate proteins in vivo, these constructs were transformed into plants by floral dip Agrobacterium mediated transformation. The yeast two hybrid (Y2H) system was employed to study protein-protein interactions. Genotypic characterization of the selected T-DNA mutant lines was carried out by genetic screening through kanamycin selection. Molecular cloning of candidate genes RFI2 and SLKs into various expression vectors was accomplished. The RFI2 expression constructs were successfully transformed into Arabidopsis plants. The overexpression construct of SLK2 and the RFI2+SLK2 double construct were successfully transformed into root callus. However, the Y2H interaction assay was inconclusive about RFI2-SLK protein interaction, but RFI2 was shown to form homodimers. The transformed plants and the tagged protein constructs of RFI2 and SLK2 will be utilized for protein purification, biochemical assay and in vitro ubiquitination assay to study protein interaction, stability and modification. The genetic screening of T-DNA lines resulted in identification of 10 homozygous lines. In phenotypic analysis of these homozygous lines, early-stage growth and developmental phenotypes such as germination, juvenile leaf emergence, rosette size, colour and root growth were observed. The phenotypic analysis is crucial in identification of the informative phenotype changes in the E3 T-DNA mutant lines. This approach has been successfully employed in understanding of genetic and molecular basis for many biological traits in plants.