The ESSA and other International Sequencing programmes of the Arabidopsis genome are projected to produce sequence information of enormous scientific and potential commercial use. For efficient use of this potential, it is essential to determine the function of novel genes and ORFs uncovered by the genomic and cDNA sequencing programmes. To establish a link between different programmes aiming at the structural and functional analysis of the Arabidopsis genome, we propose here a 'target selected gene inactivation' approach. This approach is based on the application of insertion elements, such as the T-DNA of Agrobacterium and heterologous transposons, which have been used with a great success for the isolation, mapping and functional analysis of gene mutations in Arabidopsis. Our proposal aims to achieve a saturation insertional mutagenesis of the Arabidopsis genome, in order to identify mutations in any gene of interest. The identification of mutated genes by sequence based PCR and gene fusion methods can correlate information about DNA sequence, map position and phenotype.
This strategy involves the saturation of the Arabidopsis genome with an assortment of T-DNA and transposon insertions to identify insertional mutations in target genes, the sequence of which is known.
Analogous methods have been used efficiently in higher eukaryotes carrying active endogenous transposons to complement or replace the laborious and complicated gene replacement techniques. To quickly reach genome saturation, the introduced T-DNA and transposon insertions in the Arabidopsis genome will be complemented by novel techniques, such as gametic transposition. Methods for T-DNA primed genomic sequencing and comparison with existing databases will address questions about insert distribution, integration specificity and reveal novel transcriptional regulatory sequences. Frequently occurring ESTs of unknown function will be screened by PCR for insertions using a T-DNA tagged mutant population of one genome equivalent produced by in planta transformation.
A local saturation mutagenesis strategy with mapped transposons will focus on the functional analysis of genes and ORFs identified on chromosome 4 by the ESSA programme. Novel technical solutions for the screening of inserts in any gene of interest are being developed to systematically approach genome saturation. Thus, the function of random cDNAs can be defined by gene inactivation and gene fusion technologies.
To increase the randomness of transposon insertions, a retrotransposon constructed to yield a high copy number of insertions will be used for the generation of mutations. This synergistic contribution to the ESSA Arabidopsis Genome Project should lead to the discovery of new sequences and genes of agricultural significance and facilitate their biotechnological application. In addition, the proposed action should serve as a pilot project to assess the power of advanced reverse genetics in plants, and is expected to provide a general strategy for functional characterization of Arabidopsis genes represented by genomic and cDNA sequences for use in basic and applied plant biotechnology.
Funding SchemeCSC - Cost-sharing contracts
6700 AA Wageningen
NR4 7UH Norwich
NR4 7UH Norwich