Isolation of Arabidopsis thaliana Palmitoyltransferase Mutants

1
Isolation of Arabidopsis thaliana
Palmitoyltransferase Mutants
Derek Thibault, Aaron Argyros, and Estelle Hrabak
Department of Plant Biology, University of New
Hampshire
Introduction
Plant Genotyping
Example of PCR Products Analyzed with Gel
Electrophoresis
The purpose of this study was to identify
mutations in genes encoding palmitoyl-transferases
in order to determine the functions of these
enzymes. Our model system is the plant
Arabidopsis thaliana. Palmitoyltransferases are
enzymes that catalyze protein palmitoylation.
Mutant plants will be identified by the presence
of foreign DNA insertions (T-DNA) in
palmitoyltransferase genes.
Each potential palmitoyltransferase mutant plant
was analyzed with two PCR assays
Possible Genotypes Detectable by PCR

• amplification of WT gene (red primers)
• amplification of T-DNA insert (brown primers)

• T-DNA (transferred DNA)
• Functions as an insertional mutagen
• Randomly integrated into plant genome
• 5 Kb in size

• A. thaliana
• Small fully-sequenced genome (125 Mb)
• Rapid life cycle
• Abundant seed production

• Palmitoylation
• 16 C fatty acid modification of cysteine residues
• Increases protein hydrophobicity, causing
  membrane binding

Following agarose gel electrophoresis,
presence/absence of product from each PCR allows
genotyping of plant. Examples of plants with
three different genotypes are shown at right.
Gel Electrophoresis Patterns Corresponding to
Each Genotype
Summary of Genotyping
Wild-Type
Wild-Type
Wild-Type
T-DNA
T-DNA
T-DNA
Experimental Overview
1100 bp
1100 bp
950 bp
950 bp
Homozygous
Wild-Type
Heterozygous
DNA Sequencing
DNA sequencing with fluorescent
dideoxynucleotides was used to determine the
T-DNA insertion site. An example from one
palmitoyltransferase gene is shown.
Gene Sequence From GenBank ?
ATTCCTTATCTCAGCCGTCATTAGCACAAGCTATGCTGCTGTTATGTGTG
TGTATACCTTGATTCATATCTT AAAACGTCCGCAATGCTGTTATGTGT
GTGTATACCTTGATTCATAACTT
Junction ? Sequence
T-DNA Insertion Site
Conclusions

• One or more homozygous mutant lines have been
  identified for 5 palmitoyltransferase genes.
• DNA sequencing is underway to confirm the precise
  sites of T-DNA integration.
• Experiments (data not shown) are in progress to
  determine the phenotypic properties of each
  palmitoyltransferase mutant.

Acknowledgements NSF Grant 04-29268 to E.
Hrabak