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

Functional Genomics of Stress Tolerance
307
lyzed after inoculation with the fungal pathogen Alternaria brassicicola and defenserelated
signaling molecules, i.e. salicylic acid, methyl jasmonate or ethylene, using the
Arabidopsis DNA microarrays (Schenk et al., 2000). It was found that 705 ESTs showed
differential expression in one or more of these experiments. A microarray enriched in
wound and insect responsive sequences has been used to analyze expression in
Nicotiana longiflora in response to solar ultraviolet-B radiation and Manduca sexta
herbivory elicitor (Izaguirre et al., 2003). Many photosynthesis-related genes were
down-regulated and genes involved in fatty acid metabolism were up-regulated. Interestingly,
UV-B and insect herbivory treatment similarly regulate the expression of genes
encoding a WRKY transcription factor and several other insect-responsive genes of
unknown function.
2.3.2. Oligonucleotide Microarray
Fodor et al. (1991) utilized solid-phase chemistry, photolabile protecting groups and
photolithography to chemically synthesize oligonuleotides directly onto solid substrate
for in-situ fabrication of arrays. The design of oligonucleotide sequences is
based on the information available in databases and thus there is no need to maintain a
large collection of cloned DNA molecules. To understand the response to wounding
and interaction between wounding, pathogen, abiotic stress and hormonal response,
Arabidopsis Genome GeneChip arrays (Affymetrix, Santa Clara, CA) were used (Cheong
et al., 2002). Many osmotic stress and heat shock regulated genes were highly responsive
to wounding although a number of genes involved in ethylene, jasmonic acid and
abscisic acid pathways were also activated in response to wounding. A 20-mer maize
GeneChip has also been designed from 1,500 ESTs to study differentially expressed
genes in leaf tissues infected with fungal pathogen (Simmons et al., 2002). For increasing
the specificity, 50-mer and 70-mer probe arrays have also been designed and it was
found that there were no difference between these arrays and full length cDNA arrays
(Kane et al., 2000; Ten-Bosch et al., 2001). A 25-mer oligonucleotide array (GeneChip
Rice Genome Array) representing 21,000 genes of rice cultivar Nipponbare was used to
study the gene expression during different stages of rice grain filling and to identify
genes involved in synthesis and transport of carbohydrates, proteins and fatty acids
(Zhu et al., 2003). Using the same GeneChip, Cooper et al. (2003) analyzed the expression
profile during stress response and seed development. They have found that
several genes which respond to environmental cues and stresses may also have a role
in development.
3. MUTANTS
A direct way of characterizing gene function is to mutate the gene and study the
consequence. However, effective means are not available for targeted gene replace-