ACPFG Annual Report

Technology Platforms
Whilst extensive phenotyping of the population has not been
possible with current staffing levels, it is apparent that there
are a number of mutants with altered phenotypes when
compared to wildtype material. For these lines we have begun
analyses of Ds insertion sites through generation of flanking
sequence tags.
Barley TILLING population
We generated a barley targeted induced local lesions in genome
(TILLING) population in 2007, by ethane methyl sulfonate (EMS)
mutagenesis of 10,000 barley (cv. Flagship) seeds. We sowed
500 M1 families of ten individuals, grew the plants to maturity
and documented any obvious mutant phenotypes. Seed from
individual plants was retained for further plantings and pooled
leaf tissue was taken for DNA isolation from the three healthiest
family members. Four thousand M2 families, consisting of
three individuals, were planted. DNA is being extracted from
all individuals and will be stored for characterisation of DNA
lesions. The seed that forms the mutant population is now ready
for screening.
Genome Structure Resources
We have now completed two large insert barley (var. Morex)
HindIII BAC libraries, as part of the international barley
physical mapping project. One contains 115,200 BAC
clones, with an average insert size of 115 kb, while the other
contains 153,600 clones, with an average insert size of
143 kb. Together, these two libraries cover approximately six
equivalents of the barley genome. In 2007, we produced a
BamHI BAC library with an average insert size of 120 kb. This
library contains 300,000 clones and covers seven equivalents
of the barley genome. We produced eight filter copies for
hybridisation screening of the barley HindIII BAC library, as
well assix filter copies for hybridisation screening of the rye
HindIII BAC library, and four filter copies for hybridisation
screening of the phalaris HindIII BAC library.
In addition to the barley BAC libraries constructed, we also
helped colleagues at CSIRO Plant Industry construct a lupin
BamHI BAc library. This library contains inserts around 100 kb
and has ten times genome coverage.
Promoter isolation
Promoters are DNA elements that are important in the
spatial and temporal control of gene expression in an
organism. Scientists use these promoters in the production of
constructs for plant transformation, so that the transgene can
be expressed in specific tissues, at specific developmental
stages, or in response to specific stress treatments. We isolated
promoter elements from Arabidopsis and rice enhancer trap
lines, and for Arabidopsis we were able to replicate the
patterns of cell-specific gene expression seen in the lines from
which the promoters were isolated. In addition, we isolated
promoters from rice and wheat. Five of the rice promoters
enabled drought stress inducible expression of a reporter
gene in transgenic barley, and another cold stress inducible
expression. We also modified three transformation vectors for
use in the plant transformation process.
Heterologous Expression
As a result of funding priorities for ACPFG II changing,
support for this technology platform ceased in 2007. Several
heterologous expression techniques have been implemented,
including the magnICON system developed by ICON Genetics.
A number of genes were expressed in Nicotiana benthamiana
leaf tissue using the magnICON system and were extracted and
found to be active. These included the control GFP and the cell
wall modifying enzymes EII, XEB and XTH.
Proteomics and
protein analysis
APCFG’s proteomics platform is located at the School of
Botany, University of Melbourne node. Protein abundance
and activity can be regulated through multiple mechanisms at
the level of gene transcription, translation, post translational
modification and protein degradation. In 2007, methods have
been refined to enable quantitative comparisons between
protein abundance in different tissue extracts. Robust
capabilities were established using two complementary
approaches to undertake this type of experiment; two
dimensional gel electrophoresis coupled with difference in gel
electrophoresis (DIGE) dyes and iTRAQ peptide labeling.
The iTRAQ methodology was successfully applied to the
comparison of protein abundance in B tolerant and intolerant
barley lines, and this resulted in a publication in Plant
Physiology. This approach was also applied to examining the
changes in protein abundance in the roots of barley plants
following salt stress. This study highlighted consistent changes
in proteins involved in glycolysis following extended exposure
to elevated salt, and the physiological implications of this are
being investigated.
The 2D-DIGE was used to monitor changes in protein
abundance in rice suspension culture cells, following
exposure to elevated levels of NaCl and abscisic acid. A
number of proteins with altered abundance have been
identified following these treatments. Coupled with these
studies have been the ongoing identification of proteins from
2D gels and the construction of 2D spot libraries. To date,
over 100 spots have been identified in reference 2D gels.
We also used the targeted analysis of simple protein mixtures
to identify the post-translational modification status of a
number of thioredoxin proteins. These targeted studies have
been used as a preliminary component of the antibody
production pipeline, to monitor the efficacy of protein antigen
expression levels.
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