Marker gene elimination using the Ac/Ds system in sugar beet

Topic

Future genetically modified plants should contain only foreign sequences needed for the immediate function. With the help of the Ac/Ds transposon system ("jumping genes") plants can be produced which contain only the gene of interest and which are free from additional sequences such as marker genes .

In the precursor project, the Ac/Ds system was successfully integrated into sugar beet. It was possible to verify the transposition (jumping) of the reporter gene to another site in the plant genome.

The aim of this project is to conduct further research into the frequency of transposition and integration sites on the offspring.

A further aim is to optimise the Ac/Ds system in sugar beet to produce marker-gene-free, transgenic plants within a generation. In addition, transposition in the transgenic sugar beet is to be prevented from happening more than once.

Precursor project:

• Research project: Eliminating superfluous gene sequences in sugar beet; Planta, Einbeck

Further information on the procedure and results to date:

• Jumping genes: From phenomenon to tool

Project description

In this project the offspring from the precursor project will be further examined and special transgenic sugar beet will be produced to test the optimised Ac/Ds system.

(1) Research into transposition. First the frequency of transposition in the direct offspring from the precursor project will be determined by testing the offspring for resistance to the herbicide (phosphinotricin; Ppt). This is done by dripping Ppt onto the leaves of young plants.

• If the plants are not resistant, the leaves turn white after two days and die off two days later.

• In the case of resistant plants, where transposition has successfully taken place, no changes are observed. In this instance the reporter gene has jumped to a different site in the genome during transposition, as a result of which the plant is once more able to produce the herbicide phosphinotricin. The ratio of resistant to non-resistant plants gives information about the frequency of transposition.

The integration site that the Ds element has jumped to is then analysed. It is characterised more closely using various molecular biological methods, including PCR and Southern blot analysis.

(2) Optimising the Ac/Ds system. After the first transposition, the aim is to prevent further transposition taking place in the cell. To achieve this, various vector constructs are produced, all of which contain the complete components for marker gene elimination using Ac and Ds. In addition, another gene is integrated into each of the vector constructs, the purpose of which is to severely restrict or completely stop the activity of the transposase – and to do so at the point when the first transposition of the Ds element has taken place. Transposase is the enzyme that recognises the ends of the Ds sequence and cuts the DNA at this site, thereby triggering the transposition.

On successful completion of transposition, the target and marker genes are located at different places in the plant genome. It is now possible to produce offspring from this plant that contain only the target gene and not the marker gene through natural segregation processes .

The vector constructs are tested for functionality in sugar beet and in the model plant Arabidopsis thaliana . In future the system will also be made available to other crops.

Results

Detecting Ac/Ds transposition in sugar beet: The experiments used seed from crosses and from self-fertilized transgenic sugar beet plants that had been transformed with Ac and Ds elements. In the first year of the project in 2005, segregation analyses of the sugar beet progeny were carried out on selection media. The experiments failed to shed any light on the transposition events under investigation, since the seed quality was not adequate (fungal infection) and the selection results on a medium containing antibiotics were not clear. In order to identify the plants that contained transposed elements without any additional sequences, they therefore had to be grown in soil over the course of the second year of the project (2006). Following DNA isolation, PCR analysis was used to determine the transgenic background of each plant. It was possible to identify plant lines that contained a transposed Ds element.

The analysis of transposition frequency was also to have been carried out through segregation on selection media. The poor quality of the seed meant that this project aim had to be abandoned.

To investigate the integration site, DNA was taken from the plants in which the Ds element had been detected. Using inverse PCR, it was possible to detect an integration site.

Vector constructs for optimising the Ac/Ds system: In the first year of the project (2005) a construct was produced consisting of Ac/Ds elements and the Ac transposase-regulating gene. In subsequent experiments (2006), a second construct was produced that contains a reporter gene as well (GUS gene). In the absence of a substrate, this gene colours the genetically modified cells in which the reporter gene is active. Since the reporter gene is linked to the Ac transposase-regulating gene, this means that the activity of this gene can be analysed as well. Both constructs were integrated into a T DNA vector and transformed into the model plant, Arabidopsis thaliana , using Agrobacteria . The transformation of the constructs in sugar beet plants has so far not been successful. The experiments are therefore being continued.