15th International Conference on Arabidopsis Research - TAIR

T10-033
Analysis of gene expression within single cells from
Arabidopsis thaliana
Schliep, Martin(1), Zoeller, Daniela(1), Simon-Rosin, Ulrike(1), Fisahn, Joachim(1)
1-Max-Planck Institut für Molekulare Pflanzenphysiologie, Am Mühlenberg 1, 14476 Golm,
Germany
Transcript analysis at single cell level represents a major approach in understanding
of plant complexity. The objective of the present project is focused
on temporal and spatial expression of genes within single cells.
We have optimised the single cell sampling protocol for Arabidopsis thaliana
and are able to collect routinely sap from trichome, basal and epidermal cells.
This single cell sampling technique, enabled us to analyse in a minimalinvasive
approache genes and transcripts. Samples are analysed not only
using hybridisation of nylon filter membranes, also the expression of genes
were studied by real time RT PCR experiments. Here we present exemplary
results of single cell expression study using real time RT PCR from MYB
23 (At5g40330), an transcription factor high expressed in trichomes, from
Arabidopsis thaliana.
15 th <strong>International</strong> <strong>Conference</strong> on Arabidopsis Research 2004 · Berlin
T10-034
Gene Identification by Transcript Based Cloning
James Hadfield(1), Giles Oldroyd(1)
1-john innes centre
The identification of genes required for a biological process, usually through
mutation and positional cloning, is a critical first step in identifying the molecular
players in that process. However, positional cloning can be laborious
in organisms with sequenced genomes and impractical in organisms, such
as crop plants, with large and complex genomes. We have developed a rapid
gene cloning method based on transcript abundance and present here the
identification of the Barley RAR1 gene in 1 month, as a demonstration of
this technique. Other work cloning the Medicago DMI3 gene over a 3 month
period is described elsewhere (Mitra et al PNAS 2004 101:4701-4705). We
show that Transcript Based Cloning is a viable approach for gene cloning in
crop species with large genomes.
The barley RAR1 gene is required for resistance against a range of pathogenic
powdery mildew fungi, triggered by different race-specific resistance (R)
genes. Cytological analyses have shown that two key phenotypes of racespecific
resistance, hydrogen peroxide accumulation and hypersensitive cell
death (HR) are compromised in rar1 mutants. The RAR1 gene has previously
been cloned using a map-based cloning approach that took about 5 years to
complete.
Raka M. et al A Ca2calmodulin-dependent protein kinase required for symbiotic nodule development:
Gene identification by transcript-based cloning. PNAS 2004 101: 4701-4705
T10 Novel Tools, Techniques and Resources