Targeted insertion of genes at defined sites in the oilseed rape genome

Topic

When inserting new genes in plants, it is impossible to predict where in the genome integration will occur. As a result, the newly inserted gene could impair the functioning of other genes. Targeted insertion at known and well-defined sites in the genome could eliminate this uncertainty.

The aim of this project is to develop plants which are optimally prepared for the integration of a gene at a defined location in the genome. Such precise insertion should eliminate any effects resulting from an unfavourable positioning of the newly inserted genes on the chromosomes. In addition,transgenes will be targeted directly in regions of high gene expression .

The research will be carried out on oilseed rape (Brassica napus). As well as providing oil for the food industry, oilseed rape is increasingly being used as a renewable resource in the chemical industry and for the production of biodiesel.

Experiment description

Various naturally occurring mechanisms exist for the transfer and recombination of DNA. This project will employ two of them: a transposon system and a recombination system. Transposases and recombinases are enzymes which cut out DNA segments at specific recognition sequences and then re-integrate them at a different site. Unlike transposases, recombinases also require recognition sequences for the insertion of a DNA sequence. Transposases are able to re-insert the excised DNA sequence at any location, although as a general rule they tend to integrate it in regions of high gene expression.

Both systems have already been used separately to successfully remove marker genes from plants. This project will see them combined for the first time and used for the targeted insertion of genes. A genetically modified transposon containing the recognition sequences for a recombinase will be inserted in the rape genome. It will also contain a reporter gene which will detect whether the transposon has been inserted in a region of high gene expression. Molecular-biological analysis will be carried out to check that the insertion has not resulted in any undesirable changes in the rape genome, such as the silencing of a gene. Rape strains that meet both of the above criteria will be selected as recipient strains.

Any chosen transgene can then be inserted with pinpoint accuracy in the genome of the recipient strains with the aid of a recombinase.