The Characterisation of Putative Nuclear Pore-Anchoring Proteins in Arabidopsis thaliana

• Main Author: Collins, Patrick
• Language: en
• Published: University of Canterbury. Biological Sciences 2014
• Subjects: Arabidopsis thaliana; transformation; insert; transient expression; epidermal cells; root cells; gene; protein; plant; bolting; flowering; root growth; knockout; ndc1; gp210; At1g73240; At5g40480; SALK; SAIL; Columbia; seed; germination; DNA sequencing; agarose gel electrophoresis confocal microscopy; light microscopy; stereo-fluorescence microscopy; DNA extraction; cell; nucleus; cytoplasm; nucleoplasm; nuclear pore complex; nucleoporin; nuclear envelope; putative; nuclear pore-anchoring protein; leptomycin B; gene gun; particle bombardment; agrobacterium; T-DNA; fasciation; rhodococcus fascians; cross; reverse genetics; nucleocytoplasmic transport; self; synergistic; silique; transmembrane ring; dihybrid; pumilo homology domain protein; GFP; YFP; RFP; PCR; homozygote; heterozygote; inhibition
• Online Access: http://hdl.handle.net/10092/8885

The nuclear pore complex (NPC) is perhaps the largest protein complex in the eukaryotic cell, and controls the movement of molecules across the nuclear envelope. The NPC is composed of up to 30 proteins termed nucleoporins (Nups), each grouped in different sub-complexes. The transmembrane ring sub-complex is composed of Nups responsible for anchoring the NPC to the nuclear envelope. Bioinformatic analysis has traced all major sub-complexes of the NPC back to the last eukaryotic common ancestor, meaning that the nuclear pore structure and function is conserved amongst all eukaryotes. In this study Arabidopsis T-DNA knockout lines for these genes were investigated to characterise gene function. Differences in plant growth and development were observed for the ndc1 knockout line compared to wild-type but gp210 plants showed no phenotypic differences. The double knockout line gp210 ndc1 was generated through crosses to observe plant response to the knockout of two anchoring-Nup genes. No synergistic affect from this double knockout was observed, suggesting that more, as yet unidentified Nups function the transmembrane ring in plants. The sensitivity to nuclear export inhibitor leptomycin B (LMB) was tested also for knockout lines, although growth sensitivity to the drug was not observed. Nucleocytoplasmic transport of knockout lines was measured in cells transformed by particle bombardment. To express fluorescent protein constructs actively transported through the NPC, localisation of protein determined the nucleocytoplasmic transport of the cell. The ndc1single knockout and the double knockout gp210 ndc1 exhibited decreased nuclear export. Further experiments in determining NDC1 localisation and identification of other Nups in the transmembrane ring sub-complex would bring a more comprehensive understanding to the plant NPC.