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.