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Expression and localization studies of unknown Arabidopsis thaliana genes
Author
Yasmeen Mohamad
Supervisor
Chia, Tet Fatt
Abstract
Arabidopsis thaliana is used as the model system for flowering plants because of its small genome(I25 Mb), rapid life cycle and small size. Its genome had been completely sequenced and annotated through the Arabidopsis Genome. Initiative at the end of 2000. However to date, the functions of about 4000 genes remain unknown.
In this study, two unknown genes (At2g03590 and At2gl6800) were studied and tagged with a yellow fluorescent protein gene using a modified polymerase chain reaction technique known as the triple template technique. This was done with the aim of studying the transient expression of these genes and the localization of the protein with regards to the parts of the plant and its cellular compartmentalisation. Agarose gel electrophoresis was used to confirm that the product of this amplification step is a hybrid of the unknown gene and the YFP gene. The hybrid gene was precipitated down and coated onto tungsten particles. .A gene gun was then used to shoot the genes into young A. thaliana plants of the Columbia ecotype. Expression of this gene was detected 36 hours later via inverse fluorescence microscopy and confocal microscopy.
For gene At2g03590, the triple template product could not be obtained. The results for gene At2g16800, indicated that although the gene was expressed in the leaves, petiole and roots, its level of expression was significantly higher in the leaves than in the roots (p<0.05). The tissues that were bombarded with the YFP-tagged At2gl6800 gene exhibit fluorescence throughout the cell. There is a possibility that the gene codes for a membrane protein, as suggested by the BLAST results and the hydrophobic nature of the protein. as shown by the predicted protein sequence and the hydropathy plot. Further studies with regard to the function of this gene should focus on the localization of the protein in the cellular and organelle membranes.
In this study, two unknown genes (At2g03590 and At2gl6800) were studied and tagged with a yellow fluorescent protein gene using a modified polymerase chain reaction technique known as the triple template technique. This was done with the aim of studying the transient expression of these genes and the localization of the protein with regards to the parts of the plant and its cellular compartmentalisation. Agarose gel electrophoresis was used to confirm that the product of this amplification step is a hybrid of the unknown gene and the YFP gene. The hybrid gene was precipitated down and coated onto tungsten particles. .A gene gun was then used to shoot the genes into young A. thaliana plants of the Columbia ecotype. Expression of this gene was detected 36 hours later via inverse fluorescence microscopy and confocal microscopy.
For gene At2g03590, the triple template product could not be obtained. The results for gene At2g16800, indicated that although the gene was expressed in the leaves, petiole and roots, its level of expression was significantly higher in the leaves than in the roots (p<0.05). The tissues that were bombarded with the YFP-tagged At2gl6800 gene exhibit fluorescence throughout the cell. There is a possibility that the gene codes for a membrane protein, as suggested by the BLAST results and the hydrophobic nature of the protein. as shown by the predicted protein sequence and the hydropathy plot. Further studies with regard to the function of this gene should focus on the localization of the protein in the cellular and organelle membranes.
Date Issued
2004
Call Number
QH450 Yas
Date Submitted
2004