Jun 4, 2009

Research Projects

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Obtaining marker-gene-free oilseed rape plants using the Cre/lox system

(2005 – 2008) University of Rostock, Agricultural and Environmental Faculty, Institute for Land Use (ILN)

Topic

Marker genes can be excised from transgenic plants using recombination systems. In an earlier research project, the transient Cre/lox system was successfully used in tobacco plants. In this process the Cre recombinase enzyme excises the marker gene from the plant genome. Transient means that this enzyme is active in the plant only within a specific time frame (transient expression).

The aim is to transfer the Cre/lox system to, and optimise it in oilseed rape plants in one part of the project and vines in another. A further aim of the project is to combine the recombination system with a gene of economic interest. In this project the stilbene synthase gene is used. It expresses resveratrol, a compound that is effective against fungi.

Summary

The aim of the project was to remove marker genes from oilseed rape using a temporary recombination system (Cre/loxP). The marker genes were to be eliminated by infecting the oilseed rape plants with a virus, and by using a seed-specific promoter. The experiments show that:

• the Cre/loxP recombination system was successful in removing marker genes from oilseed rape,
• when combined with a virus vector, the Cre/loxP system is not suitable for eliminating marker genes from oilseed rape,
• when combined with a seed-specific promoter, the Cre/loxP system worked in both the model plant tobacco and in oilseed rape. The progeny of these transgenic plants no longer contained the marker gene,
• the seed-specific marker gene elimination system can now be applied to other crops.

Experiment description

In oilseed rape the marker gene is transformed between two recognition sequences known as lox sites. The Cre recombinase later recognises these lox sites and excises the marker gene. This procedure should occur in the plant within a specific time frame. Two strategies are used to achieve this:

• (1) In the first approach, a virus vector is produced, which possesses the Cre recombinase gene. The gene only remains active in the cells whilst it is afflicted by the virus.
• (2) In the second approach, a seed-specific promoter is inserted in front of the Cre recombinase gene. This means that Cre recombinase is expressed only in the seeds.

Activating the Cre/lox system. In the first approach the lox-transgenic oilseed rape plants are infected with the TMV-cr-Cre virus vector. Cre recombinase is active in the infected cells and excises the marker gene. The cells are then regenerated on media containing ribavirin. Ribavirin prevents the growth of the virus.

In the second approach, progeny from the transgenic plants are produced by self-pollination. Here, Cre recombinase is active only in the seeds.

Analysis of the regenerated plants. The regenerated oilseed rape plants and the offspring resulting from self-pollination are tested for marker gene elimination and the regenerated plants are also tested to see if they are free from the virus.

Results

The project started half-way through 2005. In the first two years of the project the lox construct and the virus vector were constructed and transformed in oilseed rape plants using Agrobacteria.

(1) Virus-mediated marker gene elimination

The virus-mediated marker gene elimination method consists of two components: a TMV-cre virus vector and lox-transgenic plants.

Producing the lox constructs: The construct consists of the 35S promoter, bar herbicide resistance gene and gfp gene (reporter gene). This construct is designed to test whether the chosen strategy works in principle. The bar gene stands for the marker gene that is to be eliminated; the gfp reporter gene stands for a gene of commercial interest. PCR analysis was used to determine whether the marker and reporter genes had actually been integrated in the transgenic plants. This means that the lox cutting site is present in the plants.

Virus vector: The other component in the Cre-lox recombination system is the virus vector. However, it was only possible to infect 70 % of the rape plants with this vector. Because of these results, the vector was modified the following year (2006). The progeny of the lox plants were then infected with the adapted virus vector.

(2) Marker gene elimination using a seed-specific promoter

Two different approaches were used to eliminate the marker gene using a seed-specific promoter.

In the first, a vector was produced to ferry the lox sequences, cre gene, marker gene, promoter and the gene of economic interest for stilbene synthase into the plant in a single transformation step. The cre gene is located between the two lox sites, together with the marker gene. Excising this sequence brings the promoter close to the stilbene synthase gene and leads to seed-specific expression of the resveratrol.

The transformation vector was inserted into the oilseed rape plants via Agrobacteria. Using Southern Blot analysis it was possible to identify seven transgenic lines. These were grown in soil and then propagated using self-pollination. The progeny of these plants were then assessed for recombination events. The seeds were treated with a herbicide and their germination behaviour was observed. The majority of the lines investigated displayed sensitivity to the herbicide after three weeks. Subsequent molecular biological tests (PCR) confirmed the result: recombination had been successful in most of the lines, i.e. they no longer contained the marker gene.

In the second approach, the plants were cotransformed with two different vectors. One vector contains the marker gene flanked by lox sequences and the stilbene synthase gene. The other vector consists of the cre gene, controlled by a seed-specific promoter, and the gus reporter gene. The vectors were transferred to two different Agrobacteria strains and initially transformed in tobacco plants as a test system. 70 per cent of the lines analysed using PCR contained cre recombinase and the lox construct. These lines that tested positive were propagated using self-pollination. The seeds from the first progeny were treated with a herbicide and their germination behaviour was observed. In eight of the ten lines analysed all the plants displayed sensitivity to the herbicide, i.e. they no longer contained the marker gene. Molecular biological analyses (PCR and Southern Blot) confirmed the results.

The vectors used in this approach were then inserted into oilseed rape plants and examined using PCR for the presence of the inserted gene sequences. The vector with the cre gene had not been integrated into the oilseed rape genome of any of the plants analysed. This means that marker gene elimination using cotransformation is not suitable for oilseed rape.