Genetic engineering of Agrobacterium tumefaciens to target chloroplasts and identification of a novel nuclear localization signal of VirD2 protein

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Matsuoka, Aki
University of Lethbridge. Faculty of Arts and Science
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Lethbridge, Alta : University of Lethbridge, Dept. of Biological Sciences
Agrobacterium tumefaciens has been adopted to become a greatest tool for plant genetic engineering. Since Agrobacteria predominantly integrate single copy of the transgene into the nuclear genome, the transgene expression is relatively low. In contrast, transgene expression from chloroplasts allows high expression. Therefore, my first objective was to develop a new method of chloroplast transformation by modifying Agrobacterium genetics. I attempted to transform tobacco chloroplasts by Agrobacterium carrying the VirD2 protein with inactivated nuclear localization signals (NLSs) fused to a chloroplast transit peptide. However, the transformation of tobacco chloroplasts was unsuccessful. It suggested that the transit peptide was overridden by a novel cryptic NLS of VirD2. My second objective was to determine a novel NLS in VirD2. I discovered the novel NLS in VirD2 by subcellular localization analyses of fusion proteins (fragment of VirD2-green fluorescent protein) in plant cells. My discovery contributed to characterization of VirD2.
Agrobacterium-mediated transformation , Chloroplast transformation , VirD2 , Nuclear localization signals , Transit peptides , Protein diffusion through the nuclear pore , Subcellular localization , Protein subcellular localization in leaves