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2002
LSU-HHMI Summer Undergraduate Research Program |
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 Jason
Churchman (John C. Larkin, LSU Dept. of Biological Sciences)
The Use of Activation Tagging to Identify Genes Involved
in the Siamese Pathway in Arabidopsis thaliana
Trichomes (leaf hairs) of Arabidopsis thaliana are a good model
for the study of plant cell differentiation. Trichomes show
many aspects of plant cell growth, such as cell expansion and
specialized cell wall synthesis that are seen in other plant
cells. Trichomes are also easily visible on the cell surface
making mutant phenotypes easy to recognize.
The goal of this project is to identify new mutations affecting
trichome development using a strategy called "Activation
Tagging" (Weigel et al., 2000). The basis of this strategy
is the insertion of a plasmid at random points in the plant's
genomic DNA. This plasmid contains a strong promoter that will
activate any genes located next to the DNA insert and will result
in the over-expression of these genes. This integration of the
T-DNA immediately next to the over-expressed gene effectively
"tags" the gene since the sequence of the T-DNA is
known. This allows for the isolation of that gene and the identification
of its position on the chromosome with the use of sequencing.
The plasmid, pSKI015 (Weigel et al. ,2000) was used in this
project. This plasmid contains a basta - resistance gene and
four copies of the 35S promoter from the Cauliflower mosaic
virus (CaMV) in the T-DNA segment. Basta, the active ingredient
in many herbicides, inhibits the enzyme Glutamine Synthetase
which causes toxic levels of ammonia to buildup within the plant
cells so that only plants which have the T-DNA insert will survive.
The plasmid pSKI015 was introduced into Agrobacterium tumefaciens
bacteria via electroporation. Plants were then infected with
the Agrobacterium by dipping flowers into the cell culture medium.
Agrobacterium has the ability to transfer T -DNA segments to
plants, where they will integrate at random into the plant's
genomic DNA.
Siamese mutant Arabidopsis plants, which have multi-cellular
trichomes as opposed to uni-cellular wild-type trichomes, were
transformed with the Agrobacterium containing the pSKI015 plasmid.
Seeds from these plants were harvested, planted on soil and
sprayed with Finale, an herbicide containing basta, to select
for transformed plants. The plants that survive the application
of the herbicide will contain the T-DNA insert and will be screened
for altered trichome phenotypes. We’re interested in any
mutants that have altered trichome phenotypes since these are
good candidates to lead us to other genes involved in the Siamese
pathway. Activation tagging will allow us to easily clone and
identify these genes.
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