Physiology and Molecular Biology of Stress ... - KHAM PHA MOI

Functional Genomics of Stress Tolerance
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Expression sequence tags (ESTs) represent nucleotide sequences of small
portions of the expressed genes. These ESTs serve to tag and to fish out the putative
genes and also help quantify their expression. ESTs are also found to be useful in
understanding gene structure (Kan et al., 2001) and cloning of agronomically important
genes using ESTs based physical maps (Kurata et al., 1997). The high throughput
technology of sequencing in the last decade has helped in obtaining sequences of
partial or complete cDNA clones and their subsequent identification through sequence
homology with known ESTs or genes from other plant species or even other organisms.
ESTs served as the central resource in studies of global gene expression through highdensity
microarrays and analysis of complex traits such as drought and salinity tolerance
in Arabidopsis (Seki et al., 2001, 2002b), rice (Yamamoto and Sasaki, 1997; Kawasaki
et al., 2001) and barley (Ozturk et al., 2002). In addition to Arabidopsis and rice, large
scales EST projects are underway also for various crop species like maize (ZmDB, http:/
/www.zmdb. iastate.edu/zmdb/EST), wheat (ITEC, http://wheat.pw.usda. gov/genome/
) and sugarcane (SUCEST; http: sucest.lad.ic.unicamp.br) for general and specific traits.
In the sugarcane EST project, ‘SUCEST’, more than 2,60,000 cDNA clones were partially
sequenced from standard cDNA libraries generated from different tissues (Vettore
et al., 2003). Analysis of SUCEST database identified 88 resistant gene analogs (Rossi
et al., 2003) and 33 putative cold-regulated proteins (Nogueira et al., 2003) based on
their homology using BLAST search. Nogueira et al. (2003) found several cold inducible
genes, which have not been previously reported as being cold inducible, including
those for cellulose synthase, ABI3-interacting protein 2, OsNAC6 protein and phosphate
transporter by the analysis of around 1500 ESTs. Pih et al. (1997) identified 15 salt
stress inducible genes with early, late or continuous patterns of expression in
Arabidopsis after sequencing of 220 clones randomly from cDNA library. Bohnert et al.
(2001) have constructed more than 50 cDNA libraries from plants, bacteria and fungi for
comparative analysis of genes regulated under stress conditions.
In general, cDNA libraries represent a collection of transcripts and are likely
to miss transcripts synthesized in lower amount. For rapid identification of differentially
expressed genes, normalized or subtractive cDNA libraries (Bonaldo et al., 1996) provide
a direct mean of enriching for unique cDNA species by eliminating common
sequences. A number of highly effective methods are available to construct normalized
and subtractive libraries (Soares et al., 1994; Bonaldo et al., 1996; Diatchenko et al.,
1996; Kang et al., 1998; Carninci et al., 2000). In rice, a large number of candidate ESTs
have been found related to abiotic stress by functional annotation and subtracted
cDNA analysis (Babu et al., 2002; Sahi et al., 2003).
Functional annotation of full length cDNA in Arabidopsis (Seki et al., 2002a;
Oono et al., 2003) and rice (Kikuchi et al., 2003) as well as rapid growth of EST databases,
gene prediction and functional definition have sharply improved knowledge
about transcriptome of plants. Its correlation with various stresses would help initiate
investigation into plant stress responses.