Transposon mutagenesis in rice has recently emerged as a model system
for the study of plant functional genomics (Enoki et al. 1999,
Chin et al. 1999, Greco et al. 2000). Sorting genes from
the background of non-expressed genomic DNA remains one of the greatest
challenges in this area, and a number of techniques based on the high-throughput
detection and mapping of expressed DNA sequences have been used, such
as exon trapping, cDNA selection, reverse blotting with complex cDNA probes
and the random cloning and mapping of expressed sequence tags. We have
devised an approach called Tagged Transcriptome Display (TTD), in which
a population of rice plants carrying the maize autonomous transposable
element Ac is screened using a combination of transposon insertion
display and macroarray-based functional genomics to selectively detect
transposon insertions in transcribed sequences (Yephremov and Saedler
2000). Uniquely, we have applied this strategy to an indica rice population,
whereas previous reports have exclusively studied japonica cultivars.
We generated a population of indica rice plants carrying the Ac
transposon by introducing the transposon into rice embryos by particle
bombardment. The transposon was flanked by the maize ubiquitin-1 promoter
and the gene for green fluorescent protein (GFP). In this way, active
excision of the element could be monitored by assaying for the onset of
GFP expression (Fig. 1; Greco et al. 2000). We found that the behavior
of Ac elements in rice differed to that in maize. For example, we found
that transposition occurred very early in development, so that about 40%
of the callus derived from bombarded explants expressed GFP universally.
We also found that the element could reinsert a great distance from its
original location. In maize, the Ac element usually inserts only
a few centiMorgans away from the original site, whereas we frequently
observed genetic segregation of Ac and the gfp marker in
rice indicating long-distance transposition.
Three to four weeks after transformation, we assayed the callus for GFP
activity. Initially, we divided the callus into three groups: a) callus
with no GFP activity; b) callus with mosaic GFP activity; and c) callus
with GFP activity in all cells. PCR analysis of these tissues was then
carried out using primers spanning the junction between the promoter and
the Ac element, primers spanning the junction between the Ac
element and the gfp gene, and primers delineating just the Ac
element itself (Fig. 1). If there has been no Ac excision, the
junction-spanning primers generate a left and right full donor site (FDS)
product. However, if the Ac element has excised, the external primers
generate a single empty donor site (EDS) product (Fig. 1).
Of the callus with no GFP activity, 90% generated FDS and Ac products,
but no EDS product, showing there had been no Ac excision. The
remaining 10% failed to generate any PCR products at all, indicating the
cassette had been disrupted upon integration. Most of the callus with
mosaic GFP expression generated both FDS and EDS products, indicating
mosaic Ac excision or perhaps the presence of multiple Ac
elements, only a proportion of which had excised. Other callus in this
category generated only the EDS product, indicating universal non-mosaic
excision of the Ac element. The callus with universal GFP activity
generated only Ac and EDS products. These results were confirmed
by Southern blot hybridization to genomic DNA.
To identify transcribed sequences flanking transposon insertion sites,
was digested with SalI, which does not cut within the
Ac element. Each Ac element would thus be present embedded
within a larger genomic DNA fragment defined by SalI sites. The
products were self-ligated and amplified by PCR using inverse primers
annealing to the Ac element. Using a three-tier nested PCR strategy,
we selectively amplified only those genomic fragments directly linked
to an Ac insertion. The tertiary PCR gel, containing the third-round
nested PCR products was blotted and probed with total rice cDNA. This
identified 41 out of approximately 200 products corresponding to rice
genes. All these PCR products were subcloned in the TOPO-TA vector and
then sequenced. The sequences were compared to those in the NCBI database
revealing 41 hits on previously isolated rice and Arabidopsis genes
and ESTs. The fact that approximately one quarter of the genomic sequences
flanking the transposon insertion sites turned out to be transcribed sequences
indicates that transposition events may not be entirely random and that
there is preferential transposition into transcribed sequences.
The rice genome sequencing project is expected to uncover up to 30,000
genes, half of which will have no known function. Transposon mutagenesis
using knock-out and gene detection insertions will be a very important
tool to discover these gene functions. However, even with a small genome
such as that of Arabidopsis, knocking out every gene would entail
100,000 random insertion events. For rice, approximately four times that
number would be required. Multiple independent inserts per plant, averaging
four in many of the Ac lines, will bring down the required number
of plants. The insertional preference of Ac for genes as described
here, will reduce the required number further by a factor of 3-5. A population
of about 20-30,000 Ac plants would therefore be sufficient to recover
knock-out mutants in most genes. These populations can be produced and
then made available to research programs worldwide involved in rice functional
Enoki, H., T. Izawa, M. Kawahara, M. Komatsu, S. Koh, J. Kyozuka and K.
Shimamoto, 1999. Ac as a tool for the functional genomics of rice.
Plant J. 19: 605-613.
Chin, H.G., M.S. Choe, S.H. Lee, S.H. Park, S.H. Park, J.C. Koo, N.Y.
Kim, J.J. Lee, B.G. Oh, G.H. Yi, S.C. Kim, H.C. Choi, M.J. Cho and C.D.
Han, 1999. Molecular analysis of rice plants harboring an Ac/Ds
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Greco, R., P.B.F. Ouwerkerk, A.J.C. Taal, C. Favalli, T. Beguiristain,
P. Puigdomenech, L. Colombo, J.H.C. Hoge and A. Pereira, 2000. Early and
multiple Ac transpositions in rice generated by an adjacent strong
enhancer. Plant Mol. Biol. (in press).
Yephremov, A. and H. Saedler, 2000. Display and isolation of transposon-flanking
sequences starting from genomic DNA or RNA. Plant J. 21: 495-505.