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2005
LSU-HHMI Summer Undergraduate Research Program |
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 Monty
Aghazadeh and Norimoto Murai, Plant Pathology and Crop Physiology,
LSU Agricultural Center
Site-Directed Mutagenesis of the Neomycin Phosphotransferase
Gene from Transposon 903
When constructing binary vectors for use in plants, many different
antibiotic resistance genes can be used as selectable markers
for plant and bacterial expression. For bacterial expression,
one of the more widely used selectable markers is the 1.6 kb
Neomycin phosphotransferase (NPTII) gene from Transposon 5,
which codes for kanamycin resistance. There is a smaller, different
1.2 kb NPT gene that also confers kanamycin resistance, which
is found in Transposon 903. Despite its smaller size, this NPT
gene from Tn903 is rarely used as a selectable marker because
of the fact that 3 commonly used restriction enzyme sites are
present within the gene: XhoI, SmaI, & HindIII. These three
restriction enzyme sites can thus interfere with processes such
as cloning, where restriction enzymes are heavily depended upon.
The purpose of this study is therefore to eliminate these 3
restriction enzyme sites from the NPT gene of Tn903 and in so
doing, prepare it for use as a selectable marker in a binary
vector. In order to eliminate these restriction sites, we designed
mutagenic primers for PCR in such a way that only one of six
base recognition sequences was changed without altering the
codon usage of the affected amino acid (silent mutation). The
first step of this process was to clone the Tn903 NPT gene into
pBluescript KS-, a vector that is relatively easy to work with.
After this, the basic procedure was to perform PCR with the
1st set.
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