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Browsing by Subject "yeast two-hybrid"

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  • Molecular cloning of ARR genes and characterization of ARR proteins’ E3 ligase interaction domains 
    Zinchenko, Ganna (2016)
    The characterization of flower-specific ubiquitin-proteasome system (UPS) components and identification of their functional molecular networks will help to elucidate the involvement of UPS in regulating flower development and/or flowering time and, therefore, reproductive success of the plant. UPS component COP1 ubiquitin E3 ligase is known to regulate flowering time. The earlier data suggests that COP1 could be involved in regulating cytokinin signaling possibly through Arabidopsis Response Regulator1 (ARR1) ubiquitination. ARR1 is a B-type cytokinin response regulator, and it has recently been shown to be an unstable protein. Furthermore, KMD, F-box protein in SCF E3 ligase complex, has been shown to interact with ARR1 as well. The aim of this study is the characterization of COP1 interaction with novel target proteins ARR1, ARR2, ARR10 and ARR12 that appear to be regulated in different ways. Moreover, KMD proteins were included within the study as a possible competitor of COP1 for interaction with ARR1. In order to perform interactome studies, yeast two-hybrid assay with a preceding molecular cloning of the genes of interest was used. The results can be used to unravel the role of ubiquitin mediated regulation of cytokinin pathway.
  • Protein-protein interactions of the flavonoid biosynthetic enzymes in Arabidopsis thaliana 
    Xiang, Jiale (2014)
    Flavonoids are a group of secondary metabolites, which are not only important for plants’ survival, but also have been found to have medicinal properties for human health. Several enzymes are involved in the flavonoid biosynthesis. It is thought that these enzymes work together and may form enzymatic complexes. But the way of these enzymes interact with each other is still not clear. In arabidopsis, the number of gene family members that encode these enzymes is less than in other model plants, which makes it as a suitable model to investigate the interactions of enzymes involved in the flavonoid biosynthetic pathway. In this study, ten full-length flavonoid pathway genes were successfully amplified from cDNA of the arabidopsis flower. They are PAL1, C4H, CHS, CHI, F3H, F3’H, DFR, FLS1, ANS and GT. These genes were cloned into different prey vectors (pPR3-N and pPR3-SUC) and bait vectors (pDHB1 and pBT3-SUC). After that, the constructs were transformed separately into yeast. The protein-protein interactions were analyzed via yeast two-hybrid system.

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