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

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A.K. Tyagi, S. Vij and N. Saini
may cause transcriptional activation of nearby genes, hence this approach is referred to
as activation tagging. Weigel et al. (2000) reported a large collection of such mutants in
Arabidopsis using a T-DNA vector with four copies of CaMV 35S enhancer element.
Several different stresses were used as screens to study these mutants. In the screen
for disease resistance, two mutants were characterized at the molecular level, one of
them showing a disease resistant (cdr1-1D) and the other showing a disease sensitive
(cds1-1D) phenotype were identified.
Mutant studies provide valuable data for functional validation of gene function,
but since this is also a transgenic approach, environmental issues are naturally
raised (Hirochika, 2001). To overcome these concerns, endogenous transposons are
desirable. Tos17, a rice retrotransposon, has been used for large-scale mutagenesis
(Miyao et al., 2003). Tos17 is activated only in tissue culture conditions and is not
active under normal conditions. There are only two copies of Tos17 under normal
conditions in japonica rice and 5-30 transposed copies are found in plants regenerated
from tissue culture. A total of 47,196 lines were generated through tissue culture. Analysis
of flanking sequences from 4,316 lines showed that disease/defense related genes
constituted a total of 13.8% of the total of 16,784 independent sequences analyzed.
A number of genes have been identified through insertional mutagenesis
whose functional analysis is underway (Table 2). There are largely two strategies for
screening insertion lines generated through T-DNA and transposon tagging. One is a
‘pooling’ strategy in which ~20-100 insertion lines form a pool and these pools can be
combined to form super pools so that PCR screening can be done in stages. PCR
screening of these pools is done using gene- and insert-specific primers. An alternative
to this strategy is sequencing of regions flanking the insertion-site. Though this strategy
is definitely more time consuming, but it provides more precise information so that
one can easily search for a particular gene of interest in the database by BLASTN
searches. These flanking sequence tag (FST) databases can be made more useful by
linking them to phenotype information (Hirochika et al., 2004). These databases for
sequenced flanking insertion sites will expand in the near future and make the task of
searching the databases available for reverse genetics much easier as this will lead to
shift from chance-driven to sequence-driven screens for mutant analysis (Krysan et al.,
1999).
3.3. Traps
A significant drawback in the use of mutants for functional analysis is that the phenotype
is not always evident on mutating the gene which can happen because of functional
redundancy of the genes or because the mutation is conditional and there is no
evident morphology even in the presence of severe physiological defects (Springer,
2000; Bouche and Bouchez, 2001). To overcome such problems and complement other
efforts, traps: modified tags containing a reporter gene, have been developed. There are
three basic kind of traps, gene, enhancer and promoter traps. The expression pattern of