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

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A.K. Tyagi, S. Vij and N. Saini
3.2. Insertional Mutagenesis
Insertion of a known segment of DNA into a gene of interest is a commonly used
strategy for mutagenesis. Insertional mutagenesis offers a more rapid mean to clone a
gene as the insertion not only creates a mutation but also serves to tag the region,
which helps in its identification (Bouchez and Hofte, 1998). The two most commonly
used tools for insertional mutagenesis in plants are use of transposons or T-DNA tags
(Krysan et al., 1999). T-DNA is a segment of the tumour-inducing (Ti) plasmid of
Agrobacterium tumefaciens and is delimited by short imperfect repeat border sequences.
The T-DNA including any region between its left and right border can be transferred by
Agrobacterium into the plants where the T-DNA gets inserted in the plant genome.
The mutants are screened based on unique sequence within the left and right border,
for instance a selectable marker gene or a reporter gene (Azpiroz-Leehan and Feldmann,
1997). T-DNA tagged lines have been generated in Arabidopsis with the specific aim of
isolating mutants altered in stress signaling. These tagged lines have been generated in
the transgenic background of a stress inducible RD29A promoter (responsive to cold,
salt, dehydration and ABA) fused to luciferase reporter gene. Luciferase enzyme activity
was monitored in response to several stresses. On this basis, three groups of mutants
have been identified, cos (constitutive expression of osmotically responsive genes),
los (low expression of osmotically responsive genes) and hos (high expression of osmotically
responsive genes) (Ishitani et al., 1997). Preliminary screening of this collection
identified 43 mutants in the ‘hos’ category and 20 in the ‘los’ category (Bohnert et
al., 2001). Several important genes in abiotic stress pathway have been identified using
these mutants (Table 2). Jeon et al. (2000) developed 22,090 T-DNA tagged lines in rice
of which 18,358 were fertile. These lines have recently been used for identifying lowtemperature-responsive
genes in rice (Table 2). Lee et al. (2004) analyzed 15,586 rice
lines, out of which 81 (0.52%) showed cold-responsive GUS expression. Also, 37 tagged
genes were identified from these lines, two of which, OsRLK1 (putatitive LRR-typereceptor
like protein kinase) and OsDMKT1 (putative demethylmenaquinone methyl
transferase) were confirmed experimentally to be cold-responsive.
Transposable elements have also been used on a genome-wide scale for functional
analysis. Endogenous transposable elements have been identified and
characterized in several plant species (Ramachandran and Sundaresan, 2001).
Transposons have been used in heterologous systems also to identify gene function,
Activator/Dissociation (Ac/Ds) and Enhancer/Supressor-mutator (En/Spm) being two
most widely used transposable elements for insertional mutagenesis (Parinov and
Sundaresan, 2000). The maize En/Spm element was used for generating 48,000
Arabidopsis lines. A total of 420 tagged genes were identified based on analysis of
1,200 flanking sequences (Tissier et al., 1999). Using En/Spm elements, Speulman et al.
(1999) reported the production of 2,592 lines. Around 250 flanking sequences were
analyzed from which 100 tagged genes were identified but none of them were found to
be involved in stress response. For characterizing the MYB family of genes, three
transposon tag collections (Baumann et al., 1998; Speulman et al., 1999; Tissier et al.,