Proceedings
of the XLVI Italian Society of Agricultural Genetics - SIGA Annual Congress
Giardini
Naxos, Italy - 18/21 September, 2002
ISBN 88-900622-3-1
Poster
Abstract - 5.02
cloning of early-stress induced gene in
durum wheat via PCr-select
Mastrangelo A.M.*,**, mazzucotelli E.*,*****,
Marè C.***, barilli S.***, ruperti B.****, grossi M.***, di fonzo N.*
*)
Experimental Institute for Cereal Research, I-71100 Foggia, Italy
**) Department Genetics
and Microbiology, University of Milano, 20133 Milano, Italy
***)
Experimental Institute for Cereal Research, I-29017 Fiorenzuola d’Arda
(PC), Italy
****) Department of Environmental Agronomy and Crop Science, Univ
of Padova, I-35020 Legnaro (PD)
*****) Department of
Biology, University of Ferrara , 44100 Ferrara, Italy
cold,
drought, transcription factor, PCR-select
In a general view the cellular
response to the stress event consists of three steps, the first one being the
sensing of the stress presence at the cellular membrane level; this information
is then transferred to the nucleus by means of a phosphorilation cascade, and
here, regulatory proteins direct the expression of downstream genes whose
products mediate stress tolerance. The use of cloning techniques has led to the
isolation of a large number of genes whose expression is up-regulated by the
stress event, but the majority of them belong to the group of downstream genes,
and only few regulatory genes have been studied, so that the topic is now to understand
the early molecular events in stress sensing and signal transduction.
To this aim
suppression subtractive libraries of control and cold stressed durum wheat
samples have been constructed by using the Clontech PCR-select cDNA Subtraction
Kit. This is a unique method based on selective amplification of differentially
expressed sequences, which overcomes technical limitations of traditional
subtraction methods. The experiment has been carried out on seven days-old
plants, exposed to low temperature (3°C) for six hours.
From a first
screening, about fifty putatively positives clones were individuated and
subjected to further analysis. By Northern experiments, fourteen clones showed
to be really cold-induced or up-regulated.
These clones have
been sequenced and analysed using the FASTA, BLASTX and BLASTN programmes. Five
clones gave positive matches in database with known genes, representing two
putative transcription factors, a putative farnesylated protein (a class of
plasmalemma interacting protein
often involved in signal transduction) and three downstream
stress-related genes. Four clones found matches with bread wheat ESTs, and five
represent completely new sequences since they did not find matches, not even in
dbEST. The putative transcriptional factors belong to MYB and AP2 families. In
particular, the second one is a new stress-induced transcription factor which
has the AP2 domain, but does not belong to the CBF/DREB family.
The mRNA
accumulation of selected clones has been analysed in a time course from 6h to
10 days of low temperature treatment. Based on their expression profile the
clones can be divided in two groups: some of them showed a transient mRNA
accumulation, from 6h to 18h or 2 days of cold exposition, while other clones
correspond to mRNA accumulated for all cold treatment duration.
The transcript
accumulation of the isolated sequences has been studied also under dehydration
stress: 7 days-old plants were dehydrated for 0.5, 1.5 and 3h. Some clones
showed to be positive also to dehydration, while others are specific for
cold-induced response.
This work shows
as the method used is very useful in the isolation of cold-induced genes
involved in early phases of the stress response. In this work genes whose products
are probably involved in stress signal transduction and in the regulation of
stress-induced gene expression have been identified. Some of these transcripts
showed a very low expression level, suggesting that this method can be useful
also in the identification of rare transcripts. The finding of clones that have
never been previously isolated, not even as EST, opens new possibilities for
the isolation of novel genes, also with respect to advanced techniques like
cDNA microarrays.